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Endocrinology Vol. 142, No. 6 2427-2434
Copyright © 2001 by The Endocrine Society

ARTICLES

Promoter Function of Different Lengths of the Murine Luteinizing Hormone Receptor Gene 5'-Flanking Region in Transfected Gonadal Cells and in Transgenic Mice1

Tuula Hämäläinen, Matti Poutanen and Ilpo Huhtaniemi

Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland

Address all correspondence and requests for reprints to: Prof. Ilpo T. Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of these studies was to explore the sex- and tissue-specific expression of the LH receptor (LHR) gene. Fusion genes containing three different lengths of the 5'-flanking region of the mouse LHR gene (7.4 kb, 2.1 kb, and 173 bp), ß-globin intron, and the ß-galactosidase (ß-GAL) reporter gene were constructed. Function of these fusion genes [LHR (7.4 kb)/ß-GAL, LHR (2.1 kb)/ß-GAL, and LHR (173 bp)/ß-GAL] was studied in vitro and in vivo. ß-GAL expression was higher in transfected mouse Leydig (mLTC-1) than in granulosa (KK-1) tumor cells with all three constructs. The shortest LHR (173 bp)/ß-GAL construct showed the highest level of ß-GAL expression in both cell types. ß-GAL expression was clearly suppressed with the 2.1-kb promoter and was nearly undetectable with the 7.4-kb construct. In transgenic mice, all three constructs directed ß-GAL expression to adult Leydig cells, displaying decreasing intensity with increasing promoter length. Unexpectedly, ß-GAL expression was also found in elongating spermatids, but not in fetal Leydig cells. There was no expression in any ovarian cell type with the three constructs used, except that one of five mouse lines with the LHR (7.4 kb)/ß-GAL construct expressed ß-GAL in their thecal cells. Two lines transgenic for the 7.4- and 2.1-kb promoter constructs each directed high ß-GAL expression to the brain, with higher intensity in 7.4-kb lines. All promoters directed expression to the pituitary gland, some faintly to the adrenal gland. Northern hybridization analysis of the ß-GAL transcripts in Leydig cells revealed that the 173-bp promoter mainly gave rise to the full-length ß-GAL messenger RNA, whereas the 2.1- and 7.4-kb promoters mainly induced transcription of truncated ß-GAL messages. This suggests that the 5'-flanking region, upstream of -173, determines the formation of splice variants of the structural gene to be transcribed. The present findings in transgenic mice provide in vivo evidence for basal transcriptional activity of the first 173 bp upstream of the LHR translation initiation codon. In conclusion, the promoter function of the mouse LHR 5'-flanking region is tissue, age, and sex specific. The sequence upstream of the basal promoter determines extragonadal LHR expression as well as the alternate splicing of its message. The promoter sequences directing LHR expression to fetal Leydig cells and ovary reside outside the 7.4-kb 5'-flanking region and remain to be identified.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LH RECEPTOR (LHR) is a cell surface receptor comprising a large extracellular hormone-binding domain, a seven-transmembrane helix domain, and a short cytoplasmic tail. LHR is mainly expressed in gonads, testicular Leydig cells, and ovarian granulosa, thecal, interstitial, and luteal cells, and its function is essential for normal sexual development and reproductive function in both sexes. Recently, LHR expression has also been detected in several nongonadal human tissues, including the human placenta, nonpregnant uterus, and fetal membranes (1); fallopian tubes (2); uterine arteries (3); and rodent brain (4, 5), prostate (6), and adrenal gland (7, 8).

The developmental onset of LHR expression has mainly been examined in the rat and mouse. Messenger RNA (mRNA) encoding the extracellular domain of LHR, determined by RT-PCR, appears first, on embryonic day (e) 13.5, in testis and ovary (9). Expression of the full-length LHR mRNA begins later, coincident in both sexes with the developmental onset of LHR binding (10, 11) and LH-stimulated cAMP and testosterone production (10, 12). This occurs on e15.5 in the rat testis (13) and later on day 7 postpartum in the rat ovary (14) and on day 5 postpartum in the mouse ovary (15).

Studies of transcription initiation sites of the LHR gene have revealed species and sex differences. Transcription initiation sites in the rat ovary have been localized at bp -13, -14, -19, -33, and -48 (16, 17) upstream of the translation initiation codon. For mouse testicular LHR, the major transcription initiation site was found at -310 bp (18). Studies of human LHR have revealed a sex difference in the transcription initiation site, at -1085 bp in the testis (19) and at -2, -6, -18, -37, and -70 bp in the ovary (20). Despite differences in transcription initiation sites, several in vitro studies have revealed that the basal transcriptional activity of the LHR promoter lies within the first 173/176 nucleotides of the LHR 5'-flanking region in all species studied (20, 21, 22). Many in vitro studies have demonstrated that in addition to the 173-bp basal promoter, the more distal 5'-flanking region has inhibitory domains. Remarkably, these domains reduce the expression level of reporter genes (21, 23, 24, 25, 26, 27). Neither the rat nor the mouse LHR promoters contain TATA-like sequences in the basal 173-bp promoter, but instead, they have a GC-rich region with several Sp1 consensus sequences (21, 26, 27). Most experiments on the LHR promoter have used about 2-kb stretches of the 5'-flanking sequence, and studies on longer promoter sequences have not been reported. In our previous study, a 2.1-kb sequence of the murine LHR promoter displayed both sex- and age-specific functions, being confined to adult Leydig cells and having no activity in the ovary or fetal Leydig cells (5).

The LHR gene is transcribed in gonads into several splice variants of 1–7 kb in length. The sizes of the transcripts are largely similar in the ovary and testis, although differences occur in their relative abundance (28) and according to the functional stage (13, 29). The full-length coding sequence of LHR is 2.1 kb in size; however, there are at least three transcripts longer than 2.1 kb as well as a shorter 1.8-kb transcript. Transcription initiation site, splicing, and polyadenylation determine the final size of a mRNA. Intron sequences have been found to exist in different LHR transcripts, except for the longest (6.8/7.0-kb) transcript, which contains an extended 3'-untranslated region (30). The function of the 1.8-kb transcript is obscure but interesting; it is too short to encode full-length LHR, and it is assumed to encode the extracellular domain of the LHR. Expression of this particular form of LHR mRNA starts during ontogeny markedly earlier than the message corresponding to full-length receptor (9, 15). It is not hormonally regulated, as hCG treatment of adult rats caused no significant change in this transcript, in contrast to suppression of the 7-, 4.2-, and 2.5-kb transcripts (31). After deletion of the cytoplasmic domain of LHR, the truncated receptor cannot be transported and/or anchored to the plasma membrane; however, it has high LH/hCG binding activity (32). After destruction of mature Leydig cells in rats by ethylene dimethane sulfonate treatment, the 1.8-kb LHR transcript has been found to persist in the testis, whereas other transcripts are totally abolished until a new population of mature Leydig cells emerges (33).

In the present study, we examined the functional differences between various lengths of the murine LHR promoter, spanning the 5'-flanking region up to 7.4 kb relative to the translation initiation codon. We created three LHR/ß-galactosidase (ß-GAL) expression constructs carrying 7.4-kb, 2.1-kb, or 173-bp promoter sequences and studied their function in vitro in transfected gonadal cells and in vivo in transgenic (TG) mice. The results revealed that the locations and levels of expression as well as the sizes of the ß-GAL transcripts in Leydig cells were determined by the length of the promoter sequence. No promoter activity was found with any of the constructs in fetal Leydig cells and ovary, except in thecal cells in one of five mouse lines harboring the 7.4-kb LHR promoter/ß-GAL construct.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgene constructs and TG mouse production
The LHR/ß-GAL fusion genes contained 173-bp, 2.1-kb, and 7.4-kb fragments of the murine LHR 5'-flanking region (the first nucleotide above the translation initiation codon is -1) and the coding sequence of the Escherichia coli ß-GAL gene (3.2 kb) (34). The 7.4-kb murine LHR 5'-flanking region was the SacI/NcoI restriction fragment isolated from a mouse genomic cosmid library (18). The two shorter promoter regions, 2.1 kb and 173 bp, were EcoRI/NcoI and BalI/NcoI restriction fragments of the long LHR 5'-flanking region. The second intron (0.6 kb) of the rabbit ß-globin gene (35) was inserted between the LHR promoter and ß-GAL sequences. An additional polyadenylate sequence (0.5 kb) of the E. coli phosphoglycerate kinase gene was linked at the end of the constructs (Fig. 1Go).



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  		Figure 1. Structures of the LHR promoter/ß-galactosidase fusion genes. The 7.4-kb (SacI/NcoI), 2.1-kb (EcoRI/NcoI), and 173-bp (BalI/NcoI) fragments of 5'-flanking region of the murine LHR gene (-7400/-2100/-173–1) were fused with the 3.1-kb coding sequence of the E. coli ß-GAL gene. The rabbit ß-globin intron (0.6 kb) was inserted between these sequences, and a polyadenylation sequence (0.5 kb) from the phosphoglycerate kinase (PGK) gene of E. coli was added to the 3'-end of the constructs.

 
The creation of TG mice by microinjection of the fusion gene constructs, animal housing, matings of TG mice, and PCR analysis of genomic DNA to identify TG mice were carried out as previously described (5). The studies were approved by the Turku University ethical committee for the care and use of experimental animals.

Cell culture transfections
Murine Leydig tumor cells, mLTC-1 (36), were cultured in buffered Waymouth’s medium (Life Technologies, Inc., Paisley, Scotland, UK) with 9% horse serum, 4.5% FCS, and 50 mg/liter gentamicin. Murine granulosa tumor cells, KK-1 (37), were cultured in DMEM/Ham’s F-12 medium (1:1; Life Technologies, Inc.) with 10% FCS and 50 mg/liter gentamicin.

Six million cells were transiently cotransfected using the electroporation method (330 V, 960 µF; Bio-Rad Laboratories, Inc., Richmond, CA), either with 30 µg of one of the plasmids with different LHR/ß-GAL constructs or with amounts adjusted to equal molar contents. In addition, each transfection contained 3 µg promoter cytomegalovirus-luciferase to control for transfection efficiency. The transfected cells were cultured for 24 h, then harvested and lysed. The lysates were measured for ß-GAL and luciferase activities using a spectrophotometer and 1420 Victor Multilabel Counter (Wallac, Inc., Turku, Finland) as described by Sambrook et al. (38). Luciferase activity was used for correction of the transfection efficiency of the cells.

Isolation of seminiferous tubules
Testes were decapsulated, and seminiferous tubules were isolated in DMEM/Ham’s F-12 medium using a stereomicroscope. Leydig cells were scraped off tubule walls using sterile injection needles.

Northern hybridization
Total RNA was isolated from tissues and cells using the single step acid guanidinium thiocyanate-phenol-chloroform extraction method (39). Twenty micrograms of denatured total RNA were used for Northern hybridization analyses. The RNA was resolved on 1% denaturing agarose gel and transferred onto Hybond-N nylon membranes (Amersham Pharmacia Biotech, Aylesbury, UK). The membranes were prehybridized for 3–4 h at 64 C in solution containing 50% deionized formamide, 5 x SSC (standard saline citrate), 5 x Denhardt’s solution, 0.5% SDS, and 50 mg/liter heat-denatured calf thymus DNA. The 32P-labeled complementary RNAs corresponding to nucleotides 836-3019 of the ß-GAL-coding sequence were generated using a Riboprobe System II kit (Promega Corp., Madison, WI). Hybridizations and washings of the membranes were performed according to instructions of the membrane manufacturer. Hybridization signal was detected by autoradiography using an x-ray film (XAR 5, Eastman Kodak Co., Rochester, NY).

RT-PCR
Five micrograms of total RNA were treated with deoxyribonuclease I (Life Technologies, Inc.) and reverse transcribed using AMV reverse transcriptase (Promega Corp.) and a gene-specific antisense primer corresponding to nucleotides 806–786 (5'-GCTGGCGACCTGCGTTTCAC-3') of the coding sequence of ß-GAL. In the same tube, the reverse transcribed, single stranded, complementary DNA (cDNA) was amplified by PCR using the same antisense primer as that in the RT reaction and a sense primer corresponding to nucleotides -28 to -10 (5'-ACCGGAGCTCACACTCAGG-3') of the LHR 5'-flanking region. To discriminate between RNA and genomic DNA contamination, the primers were designed to flank the ß-globin intron. Fifteen microliters of the PCR product were loaded on 1% agarose gel for electrophoresis and transferred onto Hybond-N nylon membrane. For Southern hybridization, a cDNA probe for ß-GAL-coding sequence, designed to hybridize to a sequence between the primers used in PCR, were probed with [{alpha}-32P]deoxy-CTP (Amersham Pharmacia Biotech) using a Prime-a-Gene Labeling System kit (Promega Corp.). After hybridization and washing, the membrane was exposed to x-ray film (XAR 5, Kodak).

Rapid amplification of 5'-cDNA ends (5'-RACE)
Five micrograms of total RNA were reverse transcribed using the 5'/3'-RACE kit (Promega Corp.). A gene-specific antisense primer corresponding to nucleotides 805–786 (5'-GCTGGCGACCTGCGTTTCAC-3') of the ß-GAL-coding sequence was used in the RT reaction. After adding a homopolymeric A tail to the 3'-end of the cDNA, two nested gene-specific primers corresponding to nucleotides 590–569 (5'-TCCAGATAACTGCCGTCACTC-3') and 237–218 (5'-TCAGGAAGATCGCACTCCAG-3'), and the oligo (deoxythymidine) anchor primer were used to amplify the tailed cDNA in two consecutive PCR reactions. Fifteen microliters of the PCR product were loaded onto a 1.5% agarose gel for electrophoresis and transferred to a Hybond-N nylon membrane. For Southern hybridization, an oligo probe corresponding to nucleotides 46–29 (5'-CCCAGTCACGACGTTGTA-3') of the ß-GAL-coding sequence was end labeled with [{gamma}-32P]ATP (Amersham Pharmacia Biotech). After hybridization, the membrane was exposed to x-ray film (XAR 5, Eastman Kodak Co.).

Tissue staining and sectioning
The animals were killed by cervical dislocation. The tissues to be examined were dissected out immediately (testes were decapsulated), rinsed briefly in PBS, and fixed for 15–30 min depending on the size of the tissue in a solution of 0.2% glutaraldehyde, 0.1 mol/liter sodium phosphate buffer (pH 7.3), 5 mmol/liter EGTA, 2 mmol/liter MgCl2, and 2% formalin. The tissues were then rinsed twice for 15 min each time in rinsing solution [0.1 mol/liter sodium phosphate buffer (pH 7.3), 2 mmol/liter MgCl2, 0.1% sodium deoxycholate, and 0.2% Nonidet P-40] and stained overnight at room temperature in X-gal solution [1 g/liter 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, 0.1 mol/liter sodium phosphate buffer (pH 7.3), 2 mmol/liter MgCl2, 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 5 mmol/liter potassium ferricyanide, and 5 mmol/liter potassium ferrocyanide] (40). After staining, the tissues were washed three times in PBS and stored in 70% ethanol.

For histological study, the tissues were dehydrated, embedded in paraffin wax, and sectioned. The 5-µm-thick testicular sections were dewaxed in xylene and counterstained with eosin. Thereafter, the sections were photographed using a microscope with a digital camera (DC 100, Leica Corp., Heerbrugg, Switzerland).

Statistical analysis
The values of the independent cell culture experiments were pooled for the calculation of the mean ± SEM. The data were analyzed using the StatView 4.51 statistic program (Abacus Concepts, Inc., Berkeley, CA). Significant differences were determined using paired ANOVA/t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Translational expression
Cell culture studies with Leydig (mLTC-1) and granulosa (KK-1) cells revealed that of the three constructs, LHR (7.4 kb)/ß-GAL, LHR (2.1 kb)/ß-GAL, and LHR (173 bp)/ß-GAL, the one driven by the shortest 173-bp LHR promoter displayed the highest level of expression in both cell types (Fig 2Go). The constant 30-µg and equimolar LHR/ß-GAL plasmid concentrations revealed similar results, and hence, data for both were combined in statistical analysis. The expression level decreased along with increasing promoter size; with the 7.4-kb promoter it was almost undetectable in mLTC-1 cells, and in KK-1 cells it was about 20% of that of the short 173-bp promoter. With all promoters used, ß-GAL activity in granulosa tumor cells was less than 20% of that measured in Leydig tumor cells. The endogenous LHR expression in both cell types had been confirmed previously (5).



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  		Figure 2. Measurement of ß-GAL activity in KK-1 and mLTC-1 cells transfected with the LHR (7.2 kb/2.1 kb/173 bp)/ß-GAL fusion genes. Because of variation in absolute ß-GAL activities between individual experiments, the clearly highest ß-GAL activity, found in mLTC-1 cells with the LHR (173 bp)/ß-GAL fusion gene, was designated in each experiment as 100%. The activities in mLTC-1 and KK-1 cells with the other fusion genes were calculated as a percentage of the above. All ß-GAL activities were corrected for transfection efficiency by coexpression of promoter cytomegalovirus-luciferase (see Materials and Methods). The data are the mean ± SEM of three individual experiments with two replicates. **, P < 0.01 compared with activity of mLTC-1 cells with the 173-bp LHR promoter.

 
The in vivo findings of promoter function were in accordance with the in vitro results. The ß-GAL staining of whole tissues and their closer histological study revealed that all of the constructs used drove ß-GAL expression in TG mouse testes, especially to Leydig cells but also to elongating spermatids (Fig. 3aGo). However, the expression level of ß-GAL was different between the constructs: highest with the 173-bp promoter and lowest with the 7.4-kb promoter.



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  		Figure 3. a, Histological pictures of ß-GAL-stained adult testes of TG mice expressing the 173-bp (left), 2.1-kb (middle), and 7.4-kb (right) LHR promoter constructs. ß-GAL expression in Leydig cells (LC) is higher with the 173-bp promoter than with the 2.1- and 7.4-kb promoters. The expression in elongating spermatids (esd) is similar with all constructs. The bars in the micrographs are 50 µm. The table shows results for ß-GAL-stained histological testis samples of TG mouse lines with the different promoters. Samples were chosen from the two highest expressing TG lines of each construct. Samples, collected at different ages, were graded as -, +, ++, +++, and ++++ according to the intensity of blue staining in whole testis and in LC. b, ß-GAL expression in TG mouse brain. Expression was very high in the mice of line 4 with the 7.4-kb LHR promoter, and it was spread all over the brain. ß-GAL expression was also found in TG mice with the 2.1-kb promoter, but no expression was found with the 173-bp promoter.

 
The onset of ß-GAL expression in the testes was age and promoter dependent, appearing in Leydig cells from week 3 onward with the 173 bp and from week 4 onward with the longer promoters. The expression in seminiferous tubules was limited to some spermatogonia of neonatal testes with all constructs (not shown), disappearing thereafter for 3 weeks, and reappearing in elongating spermatids from weeks 3–5 onward. At the age of 2–3 weeks postpartum, the testes displayed no ß-GAL expression. Whereas Leydig cell expression was clearest with the 173-bp construct, no difference was found in spermatid expression between the constructs.

ß-GAL expression was not found in ovarian granulosa or luteal cells with any of the constructs. Faint endogenous ß-GAL activity was found in granulosa cells especially, as occurred in our previous study with the 2.1-kb LHR promoter (5). However, mouse line 4 with the 7.4-kb promoter displayed high expression in thecal cells of adult ovaries and in interstitial cells in neonatal ovaries (Fig. 4Go). The same TG line displayed intensive ß-GAL expression in the brain (Fig. 3bGo) and pituitary gland (not shown) and faint staining in adrenal glands (not shown). Brain expression was found in two of five lines with the 7.4-kb promoter and in two of five lines with the 2.1-kb promoter, but not with the 173-bp promoter. ß-GAL expression was found in the pituitary gland with all three constructs (not shown).



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  		Figure 4. Histological pictures of ß-GAL-stained ovaries of TG mice with 7.4-kb LHR promoter (line 4) and of nontransgenic control mice. a and b, Neonatal TG ovary, revealing that ß-GAL expression is localized in interstitial cells. c and d, Adult TG ovary, where ß-GAL was expressed in thecal cells. E and F, Ovaries from neonatal (e) and adult (f) nontransgenic mice. The faint endogenous ß-GAL staining was seen in some granulosa cells in adult nontransgenic ovary. Bars in the micrographs: a, 100 µm; b, d, and e, 50 µm; c, 450 µm; f, 350 µm.

 
Transcriptional expression
The full-length coding sequence of ß-GAL has a size of 3.2 kb, and its expression at the mRNA level was examined by Northern hybridization and RT-PCR. Northern hybridization of total RNA, isolated from TG mouse testes, revealed an interesting finding; the transcription pattern of the heterologous ß-GAL gene was totally different between the promoter constructs (Fig. 5aGo). The 7.4- and 2.1-kb promoters mainly directed expression of a truncated form (2.3 kb) of ß-GAL mRNA, although a faint full-length message (3.2 kb) was also present. By contrast, the 173-bp promoter mainly directed transcription of the full-length ß-GAL mRNA, and the truncated forms were present in lesser abundance. The TG lines included in this Northern hybridization assay showed no ß-GAL expression in brain or ovary. Northern hybridization analysis of the ß-GAL transcripts in vitro with mLTC-1 and KK-1 cells confirmed that these cells process the transgenes with different promoter lengths differently. Total RNA of KK-1 cells yielded similar two transcripts (~3.0 and 3.5 kb) with all promoters used, both of which differed in size from the full-length ß-GAL transcript. In mLTC-1 cells, the 173-bp promoter drove similar full-length transcript as in the TG testes, but transcripts of the 2.1- and 7.4-kb promoters were too weak to be detectable (Fig. 5bGo).



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  		Figure 5. a, Northern hybridization analysis of RNA samples from testes, ovaries, and brains of TG mice with the different LHR promoter constructs. All mouse lines with the 7.4- and 2.1-kb promoters displayed similar transcription patterns, preferring the truncated form (2.3 kb) with only faint full-length signal. Mouse lines with the 173-bp promoter mostly produced full-length transcript and clearly less the truncated forms. ß-GAL expression in ovaries and brains was undetectable even in 2.1-kb promoter line 2, in which ß-GAL protein expression was found in the brain. b, Northern hybridization analysis of total RNAs from transfected mLTC-1 and KK-1 cells. In addition to the 173-bp, 2.1-kb, and 7.4-kb promoters, these cells were transfected with promoterless ß-GAL construct (negative control) and with promoter simian virus-ß-galactosidase construct (positive control). mLTC cells yielded weak hybridization signals, but the full-length ß-GAL transcript can be seen with the 173-bp promoter. KK-1 cells revealed two alternatively spliced transcripts, both slightly different in size from the full-length transcript. Transcripts in KK-1 cells were the same with all promoters used, except for the promoterless and promoter simian virus-ß-galactosidase constructs.

 
Northern hybridization analysis of line 4 with the 7.4-kb promoter, displaying ß-GAL activity in thecal cells, adrenal glands, and at a high level in brain, showed that the ovarian and brain transcripts were clearly less abundant than those in the testis. In the ovary the transcript was slightly longer than the full-length ß-GAL mRNA, and in the brain the sizes were either the same as the full-length message or markedly longer (data not shown). Adrenal glands did not contain any clear transcripts. Northern hybridization analyses of the transgene mRNAs in seminiferous tubules and whole testes showed that the transcript patterns were similar in both samples. This finding demonstrated that there was no clearly identifiable Leydig cell-specific ß-GAL transcript formation, which would be undetectable or less abundant in seminiferous tubules (Fig. 6Go).



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  		Figure 6. Northern hybridization analysis of total RNA isolated from whole testis and seminiferous tubules samples. The transcription patterns differed between the constructs but were similar in each pair of tissue samples.

 
The RT-PCR findings confirmed the results of Northern hybridizations. When the specific antisense primer corresponding to nucleotides 805–786 of the ß-GAL-coding sequence was used, high levels of amplification were detected in the testis, especially with the 173-bp and 2.1-kb promoters, whereas only a faint signal was detected in ovarian samples (Fig. 7aGo). In testis samples expressing construct driven by the 7.4-kb promoter, a truncated amplification product missing about 400 nucleotides was detected. In the 2.1-kb promoter samples, two truncated (deletions of 400 and 600 nucleotides) and less abundant full-length amplification products were found, whereas testes with the 173-bp promoter displayed equal amounts of full-length and truncated products. A very faint truncated amplification product of ß-GAL, similar in all mouse lines carrying different lengths of the LHR promoter, was found in ovaries. RT-PCR results in vitro were similar to the in vivo findings with the same primers (Fig. 7bGo). In Leydig cells, the amplification products were the same as in the TG testis samples with different promoters. Likewise, in KK-1 cells the truncated 500-bp message was similar to that found in TG ovaries.



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  		Figure 7. a, Southern hybridization analysis of RT-PCR products of RNA from TG testes and ovaries. Deoxyribonuclease-treated RNAs from mice with the different TG constructs were reverse transcribed and PCR amplified using primer pairs select to flank the ß-globin intron. After intron splicing, the expected size of the amplification product was about 900 bp. Testes from 173-bp and 2.1-kb mouse lines revealed the expected full-length product. In the 173-bp lines it appeared most abundant, and in the 2.1-kb lines it was only faintly amplified. The 2.1-kb promoter produced clearly truncated products (with 400- and 600-bp deletions). Testes of 7.4-kb promoter yielded a very faint truncated amplification product with a 400-bp deletion. Ovarian samples with all promoters used amplified only a faint truncated product. Because of strong hybridization, signal with shorter exposure time is shown for TG testis samples with the 173-bp promoter lines. b, Southern hybridization analysis of RT-PCR products of transfected KK-1 and mLTC-1 cells. The pattern of amplification products in mLTC cells was similar to that found in TG testes. KK-1 cells as well as ovarian samples amplified only truncated products of similar size. Testis samples of TG mice with 173-bp and 7.4-kb promoters are presented as controls. Because of strong hybridization, signal with shorter exposure time is shown for TG testis samples with the 173-bp promoter lines.

 
5'-RACE made of testis RNA from a LHR (173 bp)/ß-GAL mouse and of testis, brain, and ovarian RNA from a LHR (7.4 kb)/ß-GAL mouse line (no. 4) with high brain and thecal cell expression showed that the amplification products followed the same pattern as the Northern hybridization and RT-PCR results (Fig. 8Go). The ß-GAL mRNA of the 173-bp promoter yielded a 5'-RACE product that was about 350 nucleotides long. These nucleotides apparently represent the ß-GAL coding sequence (235 bp), the remaining nucleotides of ß-globin after intron splicing, and nucleotides linking the promoter, intron, and ß-GAL-coding sequence together. In this case the transcription initiation site would be positioned close to the 3'-end of the LHR promoter. In contrast, the truncated and less abundant full-length mRNAs of the 7.4-kb promoter revealed 5'-RACE products, predicting that the transcription initiation sites would be localized both close to the 3'-end of the promoter and also about 150 and 350 bp upstream of this site. The brain samples with the 7.4-kb promoter expressed products corresponding to a transcription initiation site slightly upstream of the 3'-end of promoter and about 250 nucleotides downstream of it. In all interpretations of the 5'-RACE results it was assumed that the ß-globin intron had spliced out correctly, as occurred in the case of the 2.1-kb promoter that was sequenced in our earlier study (5). The ovarian mRNA could not be amplified with these primers.



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  		Figure 8. Southern hybridization of 5'-RACE products of TG testes with the 7.4-kb (line 4) and 173-bp promoters, and brain and ovary with the 7.4-kb promoter (line 4). The PCR products of 5'-RACE are different in testes with 173-bp and 7.4-kb LHR promoters. The 173-bp promoter yielded a PCR product of a size (350 bp) indicating a transcription initiation site close to the 3'-end of LHR promoter. The 7.4-kb promoter revealed a PCR product of 350 bp and two additional longer products (500 and 700 bp). Brain sample with the 7.4-kb promoter amplified two products both differing in size from those detected in testis. Ovaries with the 7.4-kb promoter displayed no amplification products.

 
Taken together, the Northern hybridization, RT-PCR, and 5'-RACE results indicated that the basal 173-bp LHR promoter drives the full-length ß-GAL transcript more effectively than the longer promoters. The full-length transcript has a transcription initiation site very close to the translation initiation site, whereas the truncated transcripts have transcription initiation sites more upstream.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies with the three LHR promoter/ß-GAL fusion genes verify in vivo the earlier in vitro findings that the basal transcriptional activity of the rat and mouse LHR promoters lies within the first 173 nucleotides upstream of the LHR-coding sequence (20, 21, 22). Our results suggest that this basal promoter controls full-length gene expression at both the transcriptional and the translational level. In vitro analysis with cultured mLTC-1 and KK-1 cells as well as in vivo studies using TG mice, indicated high ß-GAL expression of the LHR (173 bp)/ß-GAL fusion gene in LH target cells. In contrast, the longer promoters (7.4 and 2.1 kb) seem to contain regulatory elements that direct transcription of the structural gene into truncated splice variants that are not translated into functional protein. Earlier studies with serial deletions of a 2.1-kb 5'-flanking region of the rat LHR gene have demonstrated that the promoter is repressed by several domains between nucleotides -173 to -2056 (21, 24). Our results show that in addition to this repressing region, a more effective inhibitory region exists upstream of nucleotide -2056.

According to our Northern hybridization and RT-PCR data, the lower ß-GAL expression level of the longer promoter constructs was associated with alternate splicing of the transcribed message. Only the basal 173-bp promoter drove predominantly full-length mRNA transcription, which consequently allowed high ß-GAL protein expression. By contrast, the longer 2.1- and 7.4-kb promoters drove the transcription to truncated mRNA forms, which is probably why their translation into functionally active ß-GAL protein was defective. Both our earlier report and the current observation of negative regulatory elements upstream of the basal LHR promoter may thus be associated with this phenomenon of alternative splicing and not to the lower transcription rate per se.

Although the endogenous LHR gene is transcribed into multiple splice variants, their function and regulation are not known. The LHR (7.4 kb)/ß-GAL and LHR (2.1 kb)/ß-GAL genes with a long 5'-flanking sequence contain the same 173-bp sequence as the shortest constructs. However, the results with the longer promoters demonstrated that they were unable to direct ß-GAL transcription into translationally competent mRNA forms; apparently some key sequence(s) was missing in the fusion genes. The missing region(s) affecting the processing of primary transcripts could be an intronic sequence (41), a 3'-untranslated region sequence (42), or part of the coding sequence. The integration site may also affect the expression of a transgene, although this was probably not the case here because the same finding was made with multiple TG lines with the 7.4- and 2.1-kb promoters.

The promoter fragments used in the current study functioned in a tissue-, age-, and sex-specific fashion in TG mice, similar to the results from our previous study with the 2.1-kb LHR promoter (5). All of the promoters directed ß-GAL expression to the same testicular cells, i.e. adult-type Leydig cells and elongating spermatids. Only the intensity of expression between constructs differed, obviously according to differential translational activity of the different splice variants. No expression was found in fetal-type Leydig cells or in ovarian granulosa and luteal cells in vivo with any construct, even though ß-GAL expression was found in granulosa cells in vitro, albeit less abundantly than in Leydig cells.

The ß-GAL staining analysis showed different brain expression between the promoters. No expression was detected in four TG mouse lines with the 173-bp promoter, the 2.1-kb promoter revealed fair expression in two of the five mouse lines, and very high ß-GAL expression was found in two of the five 7.4-kb promoter mouse lines, indicating that the longer promoter drives the expression with higher intensity to the brain. There is no obvious reason why ß-GAL expression in the brain was found only in some of the TG lines with the same 7.4-kb promoter; although the integration site of the fusion gene could be the explanation. Northern hybridization analysis of the transcription pattern of brain samples with the 7.4-kb promoter (line 4) revealed that the expression level was much lower than that in the testis.

The fact that in TG mice we could not find ß-GAL expression in ovarian granulosa or luteal cells with any of the promoters used, except for thecal cells in one of five lines with the 7.4-kb promoter, provides further evidence for sex specificity of the LHR promoter function (5). All of the transgene transcripts formed differed in size from the full-length ß-GAL transcript, as confirmed further by RT-PCR, which amplified only truncated PCR products. Moreover, translation of these messages into functionally active ß-GAL could not be demonstrated. The coding sequences under the same promoter have been found to express differently spliced transcripts in different tissues (43, 44, 45). For this reason the different ß-GAL mRNA splice variants could be translationally silent, as different mRNA splicing can change the reading frame of translation.

The 5'-RACE data on testis, brain, and ovary samples leads to the conclusion that the full-length and alternatively spliced transgene mRNAs used specific transcription initiation sites. The truncated ß-GAL transcripts mostly originated from sequences upstream of the basal 173-bp LHR promoter. A similar observation was made with the GnRH gene, which is expressed in both hypothalamus and gonads. This gene is alternatively spliced in both tissues, displaying longer 5'-untranslated region of mRNAs in gonads than in hypothalamus (46). The reason why no visible 5'-RACE amplification products were produced by the ovarian sample was probably because of the weak expression level or spliced out mRNA sequences corresponding to the primers used.

These findings of tissue, age, and sex specificity of promoter function of the mouse LHR 5'-flanking region as well as the findings that there are elements upstream of the basal promoter that determine the alternate mRNA splicing prompt the identification of the component(s) affecting this phenomenon. Some additional regions of the LHR gene could be included in new fusion genes to construct a mouse LHR promoter that would operate in the same way as the endogenous LHR promoter. Identification of the promoter elements directing LHR expression to the fetal Leydig cells and ovary remains a particular challenge for future experiments.


   Acknowledgments
 
Transgenic mice were produced at the Transgenic Mouse Core Facility, University of Turku. We thank Ms. Nina Messner and Ms. Riikka Kytömaa for skilful technical assistance with the transgenic mouse production and cell cultures, respectively.


   Footnotes
 
1 This work was supported by grants from the Academy of Finland and the Sigrid Jusélius Foundation. Back

Received August 15, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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