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Endocrinology Vol. 143, No. 10 4096-4103
Copyright © 2002 by The Endocrine Society

ARTICLE

Transgenic Mice Harboring Murine Luteinizing Hormone Receptor Promoter/ß-Galactosidase Fusion Genes: Different Structural and Hormonal Requirements of Expression in the Testis, Ovary, and Adrenal Gland

Tuula Hämäläinen, Jukka Kero, Matti Poutanen and Ilpo Huhtaniemi

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

Address all correspondence and requests for reprints to: Professor 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
 
In vivo regulation of the LH receptor (LHR) promoter was studied using transgenic (TG) mice harboring fusion genes containing three different lengths of the LHR promoter (7.4 kb, 2.1 kb, and 173 bp), fused with coding sequence of the Escherichia coli ß-galactosidase (ß-GAL) reporter gene. The length of the LHR promoter significantly affected the pattern of ß-GAL expression. In the testis the shortest promoter directed expression primarily of the full-length ß-GAL mRNA, but mainly truncated messages were transcribed from the longer LHR promoter/ß-GAL constructs. The case was reversed in the ovary and adrenal gland. Furthermore, we have recently detected strong LHR expression in the adrenal gland of female mice with chronically elevated serum LH. Therefore, the regulation of the adrenal LHR expression was addressed in the present study using the LHR/ß-GAL TG mice. Elevated LH levels were achieved in the LHR/ß-GAL mice either by gonadectomy or cross-breeding them with TG mice overexpressing a chimeric protein of bovine LH ß-subunit and the C-terminal fragment of human chorionic gonadotropin-ß. In both models, ß-GAL mRNA was found in the adrenal cortex when the 7.4-kb LHR promoter was applied but not in mice carrying the 173-bp LHR promoter. The 7.4-kb construct was activated also in the ovaries in the double TG LHR(ß-GAL)/bovine LH ß-subunit/C-terminal fragment of human chorionic gonadotropin-ßmice in some theca-interstitial cells surrounding the follicles. Hence, the LHR promoter elements essential for directing ß-GAL expression to the adrenal gland and ovary (7.4 kb) are different from those recently shown to be essential for the testicular expression (173 bp). In conclusion, elevated serum LH concentrations were found seminal for the LHR promoter activation in the ovaries and adrenals, and different lengths of the promoter are responsible for reporter gene expression in the testis, ovary, and adrenal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PITUITARY GONADOTROPIN, LH, regulates ovarian and testicular function by binding to a specific cell membrane receptor, i.e. the LH receptor (LHR), in its target cells. LHR is a glycoprotein with a long extracellular, hormone-binding domain, a seven-transmembrane domain, and a short intracellular tail. LHR is mainly expressed in gonads; testicular Leydig cells; and ovarian granulosa, thecal, interstitial, and luteal cells. Recently the expression of LHR has also been found in a number of extragonadal tissues (1, 2, 3, 4, 5, 6, 7, 8), but the function of this extragonadal expression remains obscure. Chronically elevated LH levels were found to induce functionally significant expression of the LHR in adrenal glands (8, 9), although high levels of LH usually down-regulate the LHR expression in gonads (10). Obviously, the regulation of adrenal LHR expression differs in this respect from that of gonads.

The LHR gene is transcribed into a number of mRNA splice variants;their function is still not clear. RT-PCR amplification of the extracellular domain of the LHR mRNA yields a positive result at the same embryonic age, embryonic d 13.5, in the rat testis and ovary (11). The full-length LHR mRNA can be amplified on embryonic d 15.5 in the rat testis (12) but only on postnatal d 7 in the rat ovary (13) and on d 5 in the mouse ovary (14), in correlation with the onset of LH stimulation of gonadal function (15, 16, 17). So far, no function has been confirmed to the variant consisting of only the extracellular part of the LHR, and if it exists, it must be different from that of the full-length receptor. The truncated extracellular LHR domain alone is able to bind LH (18, 19, 20) but not to incorporate into the plasma membrane or to couple to the known signal transduction cascades of LH action. Also, tissue-specific features have been shown in the alternative splicing of the LHR messages (21, 22, 23).

The promoter function of the LHR has been mainly studied in vitro. The first 173/176 bp of the LHR 5'-flanking region have been found to act as the basic promoter with ability to drive expression of reporter genes in transfected cells (24, 25, 26). The expression level of reporter genes has been found to decline along with extension of the LHR promoter sequence, compared with the basic promoter (23, 25, 27, 28, 29, 30, 31). This indicates that some inhibitory domains exist upstream of the basic promoter. The same inhibitory effect of longer promoters was also found in vivo in studies on transgenic (TG) mice carrying either the basic (173 bp) or two longer (2.1 kb and 7.4 kb) LHR promoters fused with ß-galactosidase (ß-GAL) (23). Interestingly, the basal 173-bp promoter drove only expression of the full-length mRNA and the longer promoter mainly truncated forms, whereas the overall level of ß-GAL transcription was roughly similar with all promoter constructs (23). Hence, it appeared that the apparent lower ß-GAL expression with the longer promoter constructs was mainly caused by alternate splicing of the transcribed ß-GAL messages.

In the present work, we extended our studies on the LHR promoter function using the previously produced TG mouse models with the different lengths of LHR promoter/ß-GAL constructs. Besides gonadal LHR/ß-GAL expression, we paid special attention to LHR promoter function in the adrenal gland, which becomes particularly prominent in conditions with elevated LH levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgene constructs and TG mouse production
We have previously described the production of the TG mice carrying three different lengths (173 bp, 2.1 kb, and 7.4 kb) of the 5'-flanking region of the mouse LHR, also used in the present study (23). In these mice, the promoter fragments were fused with coding sequence of Escherichia coli ß-GAL, and the second intron of rabbit ß-globin gene was inserted between promoter and ß-GAL coding sequences. A polyadenylation sequence of the phosphoglycerate kinase gene of E. coli was added at the end of the constructs (5). Another TG mouse line used in the present study was that overexpressing LH, the bovine LH ß-subunit/C-terminal fragment of human chorionic gonadotropin-ß (bLHß-CTP). These mice harbor a transgene encoding the bovine LHß subunit fused with the coding sequence for the 24-amino C-terminal peptide of human chorionic gonadotropin-ß under control of the bovine LH {alpha}-subunit promoter (32). The TG mice were genotyped by PCR of genomic DNA. DNA was isolated and primers selected as described earlier (5). Animal housing and mating of TG mice followed previously described manner (5). The studies were approved by the Turku University Ethical Committee for the care and use of experimental animals.

Two lines of the LHR(7.4 kb)/ß-GAL and LHR(173 bp)/ß-GAL mice were cross-bred with bLHß-CTP mice, and double-TG mice were genotyped by using specific primer pairs for each transgene. Only female double TG LHR(ß-GAL)/bLHß-CTP mice were studied because clearly elevated serum LH was found only in females, apparently because of low activity of the bovine {alpha}-subunit promoter in male mice (33). Double-TG females were killed at the age of 6 months, and ovaries and adrenal glands were collected for ß-GAL staining and RNA isolation. Male and female LHR/ß-GAL mice were gonadectomized at the age of 4 wk and killed 3 or 6 months after gonadectomy, and their adrenal glands were collected for ß-GAL staining and RNA isolation. Isolated RNA was used for Northern hybridization and RT-PCR analyses. Testicular RNA from intact ß-GAL mice was used as control.

Culture of adrenal tumor cells (C{alpha}1) and RNA isolation
The C{alpha}1 cells were derived from an adrenal tumor of a castrated TG mouse carrying the Simian virus 40 T antigen under the murine inhibin {alpha}-subunit promoter (34). The cells were cultured in DMEM/Ham’s F-12 medium (1:1, Life Technologies, Inc., Paisley, UK) with 10% fetal calf serum and 50 mg/liter gentamicin (Biological Industries, K:B, Hemeek, Israel). Total RNA for 5'-rapid amplification of cDNA ends (RACE) analysis was isolated from cells by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (35).

RT-PCR/Southern hybridization and 5'-RACE/ Southern hybridization
For RT-PCR, 1 µg total RNA was DNase-treated with deoxyribonuclease I (Life Technologies, Inc.) and reverse transcribed using the avian myeloblatosis virus reverse transcriptase (Promega Corp., Madison, WI). Two sense primers corresponding to nucleotides -227/-207 and -28/-10 of the LHR 5'-flanking sequence and two antisense primers corresponding to nucleotides 807/786 and 237/218 of the ß-GAL coding sequence were used. The primer pairs were designated to flank the ß-globin intron to eliminate contamination by genomic DNA. For 5'-RACE analysis of endogenous LHR, 5 µg total RNA were reverse transcribed using a gene-specific antisense primer corresponding to nucleotides 975/954 of the LHR coding sequence. After adding a homopolymeric A tail to the 3'-end of the cDNA, two nested gene-specific primers corresponding to nucleotides 279/258 and 132/110 of the LHR coding sequence and the oligo (deoxythymidine) anchor primer were used to amplify the tailed cDNA in two consecutive PCR.

Fifteen microliters RT-PCR or 5'-RACE PCR products were loaded on 1–2% agarose gel for electrophoresis. Because the specific PCR product was often difficult to identify in agarose gels, Southern hybridization was carried out each time to identify and confirm the correct amplification products. For Southern hybridization, the cDNAs in agarose gels were transferred onto Hybond-N nylon membrane (Amersham Pharmacia Biotech, Aylesbury, UK) and nested cDNA probes for the ß-GAL or LHR coding sequences were designed to hybridize to the amplified PCR sequences. The cDNAs were labeled with [{alpha}-32P]deoxy-CTP (Amersham Pharmacia Biotech) using a Prime-a-Gene labeling system kit (Promega Corp.). After hybridization and washing, the membranes were exposed to x-ray film (XAR 5, Kodak, Rochester, NY).

Histological samples
The mice were killed by cervical dislocation. For ß-GAL staining the tissues examined were rinsed briefly in PBS, fixed, and stained en bloc in X-GAL solution as described earlier (5). For histological study, the tissues were dehydrated, embedded in paraffin wax, and sectioned. The 5-µm sections were counterstained with eosin and photographed using a microscope fitted with a digital camera (DC 100, Leica Corp., Heerbrugg, Switzerland).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the present study on the LHR promoter function, we directed special attention to LHR expression in the adrenal gland and ovary in LH overexpressing LHR/ß-GAL mice. Endogenous LHR has been found to express in human adrenal cortex (7), but we have been unable to demonstrate LHR expression in normal mouse adrenal gland. However, the mouse adrenal gland appears to develop LHR expression when serum LH levels are chronically elevated (8, 9). Prompted by these findings, we induced elevated LH levels in the LHR/ß-GAL mice by gonadectomy before puberty or cross-bred them with LH overexpressing (bLHß-CTP) TG mice. Because there are differences between the ovary and testis in function of the LHR gene, it was of particular interest to explore the type of expression in the adrenal gland, whether ovarian or testicular.

The adrenal glands and ovaries of the LH overexpressing LHR(ß-GAL)/bLHß-CTP double-TG female mice did not reveal detectable amounts of ß-GAL mRNA on Northern hybridization analysis. Only the testis samples of intact LHR/ß-GAL mice revealed ß-GAL transcripts in Northern hybridization; a full-length ß-GAL transcript with the 173-bp LHR promoter and several truncated transcripts with the longer LHR promoters (results not shown), as found in our previous studies also (23). The more sensitive RT-PCR/Southern hybridization method revealed transcripts in ovaries and adrenal glands of the double-TG LHR(ß-GAL)/bLHß-CTP mice, which were not found in the single-positive LHR/ß-GAL mice. Both tissues revealed full-length ß-GAL transcripts with the 7.4-kb LHR promoter, but none with the short 173-bp promoter (Fig 1AGo). The results were opposite to those on the testis, which yielded full-length product only with the shortest LHR promoter and only marginal amplification products with the long promoter. Figure 1BGo demonstrates the results of RT-PCR/Southern hybridization in the testis, ovary, and adrenal gland of intact LHR/ß-GAL TG mice. Testicular RNA with the 173-bp promoter revealed ß-GAL amplification products corresponding to the full-length as well as truncated transcripts, but RNA samples with the longer promoters (2.1 and 7.4 kb) revealed almost exclusively truncated products. With all three promoters, the ovarian RNA formed truncated ß-GAL products at very low intensity similar to that found in the testis for the 7.4-kb promoter. The adrenal samples were negative with all constructs with different promoter lengths.



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  		Figure 1. RT-PCR/Southern hybridization analysis of LHR/ß-GAL transgene expression in the testis, ovary, and adrenal gland. A, Analysis on total ovarian and adrenal RNA of LH overexpressing double-TG, LHR(ß-GAL)/bLHß-CTP mice, carrying the 173-bp or 7.4-kb LHR promoters; testis RNA was from intact LHR/ß-GAL mice. DNase-treated RNAs were reverse transcribed, and two different primer pairs were used in PCR amplification, as specified by the scheme in the upper part of the figure. The sense primer (pLHRp1) was the same in both reactions, corresponding to the 3'-end of the LHR promoter. The two specific antisense primers (ßGALp1, ßGALp2) were located in the coding sequence of ß-GAL. RT-PCR/Southern hybridization analysis revealed ß-GAL products from adrenal and ovarian RNAs with the 7.4-kb LHR promoter of double-TG LHR(ß-GAL)/bLHß-CTP mice. By contrast, testicular RNA samples isolated from intact LHR/ß-GAL mice yielded clear ß-GAL amplification products only in mice carrying the 173-bp LHR promoter. The 900-bp amplicon in 7.4-kb ovarian lane may represent an antifactual manifold of the correct reaction product. B, ß-GAL mRNA amplification from intact LHR/ß-GAL mice. Testis RNA of mice expressing the 173-bp LHR promoter construct yielded a full-length PCR product, and those expressing the longer promoters (2.1 and 7.4 kb) amplified mostly truncated messages. All promoter constructs displayed marginal activity in the ovary, with amplification only of truncated messages. No promoter activity could be seen in any of the adrenal gland (adr.gl.) samples.

 
The findings on gonadectomized female LHR/ß-GAL mice confirmed the RT-PCR results of LH overexpressing LHR(ß-GAL)/bLHß-CTP double-TG mice; adrenal glands with the 2.1- and 7.4-kb promoter constructs revealed full-length ß-GAL amplification products, whereas those from animals harboring the 173-bp promoter revealed no amplification products (Fig 2Go).



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  		Figure 2. RT-PCR/Southern hybridization analysis of LHR/ß-GAL transgene expression in adrenal glands of gonadectomized LHR/ß-GAL female mice. Total RNAs from adrenal glands were DNase treated, reverse transcribed, and amplified with the primer pairs described in Fig. 1Go. RNA samples of mice with the 2.1- and 7.4-kb LHR promoters revealed amplification products corresponding to the full-length ß-GAL transcript, but no signal was detected in RNA from mice with the 173-bp promoter construct.

 
In our previous studies, we concentrated on identification of transcription initiation sites mainly in the testis because with the method used previously, no significant ß-GAL expression was found in the ovary and adrenal gland (5, 23). Although in the present study we were able to detect reporter gene expression in the adrenals and ovaries in gonadectomized LHR/ß-GAL mice and LHR(ß-GAL)/bLHß-CTP double-TG mice, the amount of ß-GAL mRNA was still too low to allow successful 5'-RACE analysis. To obtain more information about the sites of transcription initiation of ß-GAL in the different tissues, we carried out RT-PCR analysis with a primer -227/-207 (pLHRp2) lying outside of the basic (173 bp) promoter. The antisense primer used, +237/+218 (ßGALp1), lies in the ß-GAL coding sequence. The analyses on testicular RNA of intact LHR/ß-GAL mice and ovarian and adrenal RNAs of LHR(ß-GAL)/bLHß-CTP double-TG mice revealed differences in transcription initiation sites of the three tissues. As expected, tissues of the mice carrying the shortest (173 bp) promoter did not yield RT-PCR products with the sense primer outside the basic promoter (Fig. 3Go). RNA obtained from ovaries and adrenal glands of LHR(ß-GAL)/bLHß-CTP mice, carrying the 7.4-kb promoter, however, revealed an amplification product with these primers. By contrast, the testis RNA of intact LHR/ß-GAL mice, carrying the 7.4-kb promoter, displayed no amplification products with primer pair used (Fig. 3Go). Thus, it can be concluded that at least a fraction of the ovarian and adrenal ß-GAL transcripts in the double-TG LHR(ß-GAL)/bLHß-CTP female mice have transcription initiation sites upstream of the minimal LHR promoter, which in turn is functional in the testis of intact LHR/ß-GAL mice.



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  		Figure 3. RT-PCR/Southern hybridization analysis of LH overexpressing LHR(ß-GAL)/bLHß-CTP double-TG mice. Total RNA was treated by DNase, reverse transcribed, and amplified using the pair of primers described in the scheme in the upper part of the figure. The sense primer (pLHRp2) corresponded to nucleotides -227/-207 of the LHR 5'-flanking region, and the antisense primer (ßGALp1) was located in the coding sequence of ß-GAL. The ovary and adrenal gland revealed amplification products with the primer pair. In contrast, no amplification products were observed on testis samples of single-TG mice.

 
The differences found in the initiation sites of ß-GAL constructs directed our interest toward the putative differences in transcription initiation sites of the endogenous LHR gene in gonads and adrenal glands. Although we did not succeed in producing 5'-RACE products for the reporter gene from the ovarian and adrenal samples of the LHR(ß-GAL) or LHR(ß-GAL)/bLHß-CTP females, we were able to generate 5'-RACE products for the endogenous LHR from the ovary and testis. However, 5'-RACE amplification products were missing in adrenal RNA specimens, and hence LHR expressing murine adrenal tumor cells (C{alpha}1) (34) were used. The results indicated similar transcription initiation sites among the three tissues. However, the differences in intensities of the amplification products indicated tissue specificity in the preferred transcription start sites (Fig. 4Go). The ovary and testis displayed transcription initiation sites close to the ATG codon, which was not evidently used in the C{alpha}1 cells. Furthermore, a site about 130 bp upstream thereof was used in all tissues. The ovary and adrenal tumor cells demonstrated an additional transcription initiation site further upstream of the LHR promoter, about 200 bp from the ATG codon, and this seemed to be the main transcription initiation site in C{alpha}1 cells (Fig. 4Go). These findings correlate with those presented in Fig. 3Go.



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  		Figure 4. 5'-RACE/Southern hybridization analysis of endogenous LHR expression in ovarian and testicular RNA of non-TG mice and RNA of C{alpha}1 cells. The transcription initiation sites of endogenous LHR in the testis were located near the ATG codon and about 130 bp upstream of that site in LHR 5'-flanking region; an additional site at -200 bp was found in the ovary. C{alpha}1 cells revealed a transcription initiation site mainly at bp -200.

 
In line with the RT-PCR results, histological analysis of double-TG LHR(ß-GAL)/bLHß-CTP adrenal glands displayed ß-GAL staining in animals with the 7.4-kb LHR promoter but not with the 173-bp promoter (Fig. 5AGo). The most intense ß-GAL staining was found in zona glomerulosa and zona fasciculata. The expression was focal, with only a part of the cortex displaying cells intensively blue stained for ß-GAL. No ß-GAL expression was found in the adrenal medulla. ß-GAL staining in the adrenal glands of gonadectomized LHR/ß-GAL mice showed reporter gene activity with the 7.4-kb promoter but not with the 173-bp promoter (Fig. 5BGo). After gonadectomy (3 and 6 months), the adrenal ß-GAL staining was fairly weak, compared with the intense staining in double-TG LHR(ß-GAL)/bLHß-CTP mice. No ß-GAL expression was found in the adrenal medulla of gonadectomized LHR/ß-GAL mice.



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  		Figure 5. A, Histological pictures of ß-GAL-stained adrenal glands of LHR(ß-GAL)/bLHß-CTP double-TG mice. ß-GAL protein was visible in adrenal glands of mice with the 7.4-kb LHR promoter (b, c), but those from mice with the 173-bp promoter (a) were negative. The spotty ß-GAL staining was located mostly in zona glomerulosa and zona fasciculata in radial fashion. Adrenal medulla was negative. ZG, Zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; m, medulla. Bars: a, 20 µm; b, 40 µm;c, 6 µm. B, Histological pictures of ß-GAL stained adrenal glands of gonadectomized LHR/ß-GAL mice. Adrenal glands were stained 3 and 6 months after gonadectomy. Blue ß-GAL staining was located mostly in zona reticularis of adrenal cortex (arrows) with the 7.4-kb promoter (d, e, f). ß-GAL staining was not found in adrenal glands of mice with the 173-bp promoter (a, b, c). ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; m, medulla. Bars: a, d, f, 20 µm; b, e, 10 µm; c, 40 µm.

 
Histological analysis of ß-GAL-stained double-TG LHR(ß-GAL)/bLHß-CTP ovaries revealed ß-GAL expression in mice with the 7.4-kb ß-GAL promoter. No expression was found in granulosa cells, but it was localized to the hypertrophic theca-interstitium. No ß-GAL expression was seen in the 173-bp ovaries (Fig 6Go).



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  		Figure 6. Histological pictures of ß-GAL-stained ovaries of LHR(ß-GAL)/bLHß-CTP double-TG mice with 173-bp (a) and 7.4-kb (b through e) LHR promoters. Ovaries were stained at the age of 6 months. Strong ß-GAL staining was found in theca-interstitium with the 7.4-kb promoter, but the ovaries from mice with the 173-bp promoter were negative. Bars: a, b, 80 µm; c, 40 µm; d, 20 µm;e, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have reported previously that the ß-GAL fusion genes driven by the 173-bp, 2.1-kb, and 7.4-kb LHR promoters displayed in vitro and in vivo different tissue specificities of expression (23). In the present study, we obtained further evidence for differences in the regulation of LHR promoter in the ovary, testis, and adrenal gland. Chronic elevation of serum LH was found to have qualitative and quantitative effects on LHR promoter activity, and besides the gonads, reporter gene expression was also induced in the adrenal cortex. The mouse adrenal cortex differs from that of most other mammalian species by the presence of the elusive X zone located in its innermost region. It differentiates postnatally and degenerates in males after puberty and in females after the first pregnancy (36). In both sexes, the zone persists after gonadectomy (37, 38), which indicates the existence of a direct or indirect gonad-adrenal link. Indeed, LHR expression has been demonstrated in human and mouse adrenal glands (7, 8, 9). Human adrenal glands, collected postmortem from men and women with normal LH secretion and without adrenal diseases, displayed LH/human chorionic gonadotropin receptor protein and transcripts in zona reticularis and the deeper region of zona fasciculata (7). The mouse adrenal gland, upon chronically elevated serum LH, expresses LHR transcripts radially across the whole cortex (8). The differences in location of the LHR expression in human and mouse adrenal gland could be due to species differences in the zonation or because high LH levels are required for the ectopic LHR expression in mice.

Similar to that found for the endogenous LHR expression of the single-TG bLHß-CTP mice (Kero, J., M. Mikola, J. Nilson, R. Keri, M. Poutanen, and I. Huhtaniemi, unpublished data), we detected ß-GAL expression in LHR(7.4-kb)ß-GAL/bLHß-CTP double-TG mice in adrenal zona glomerulosa and zona fasciculata. However, LHR expression was also found in zona reticularis of the bLHß-CTP mice. In contrast, when elevated LH levels were induced in the LHR(7.4-kb)/ß-GAL mice by gonadectomy, ß-GAL expression was localized mainly to zona reticularis. In all cases, ß-GAL and LHR expression was confined to the adrenal cortex. Hence, the zonal localization of the LHR promoter function, whether monitored by transgenic ß-GAL or endogenous LHR expression, displayed some variability, depending on the way the elevation of LH secretion was brought about. The LH level is about 15 times higher in bLHß-CTP females, compared with control female mice, and in the case of gonadectomized females, the relative LH elevation is even higher (8). Because the LHR expression was weaker in adrenal glands of the gonadectomized mice, compared with the bLHß-CTP mice, it is apparent that some additional factors dependent on the presence of intact gonads synergize in the up-regulation of adrenal LHR expression. This aspect is discussed in more detail below.

At the moment the information on hormonal regulation of the complex alternative splicing of LHR mRNA is limited. However, the germ cell stage-dependent variation of LHR transcripts has been identified in both rat ovary and testis (39, 40). In the rat ovary, four LHR transcripts were detected during most of the stages of follicular development (5.8, 4.4, 2.6, and 2.3 kb in size), whereas three other forms (8.0, 1.9, and 1.4 kb) were present only in certain developmental stages (39). In the rat testis, only 1.8- and 4.2-kb mRNA transcripts were found to be present at the age of 5 postnatal d, and additional 7.0- and 1.2-kb transcripts appeared at the ages of 10 and 15 d, respectively. From d 25 onward, all mRNA transcripts were present (40). The three LHR mRNA isoforms detected in turkey ovary were observed to express at different levels during follicular development (21, 22). On the other hand, prolactin (PRL) treatment changed the steady-state levels of these LHR mRNA isoforms in theca cells so that the amount of two transcripts increased and one transcript was not altered during treatment (21). The influence of PRL on LHR induction also has been addressed in other studies, but the action of this hormone is complicated by its bidirectional regulatory effects (41, 42). Our previous studies with the bLHß-CTP mice indicated that even if high concentrations of LH are a prerequisite for the induction of adrenal LHR expression, functionally significant levels of this receptor are induced only in the presence of ovaries, probably through the estrogen-stimulated increase in circulating PRL levels (8). Hence, the induction of adrenal LHR is likely to represent a combined response to elevated LH and PRL. There is ample evidence in rodents about the stimulating role of PRL in gonadal LHR expression (43, 44).

In the present study, we found differences in adrenal ß-GAL expression between the gonadectomized LHR/ß-GAL mice and the intact LHR(ß-GAL)/bLHß-CTP double-TG mice; the expression was clearly lower in the former model. Hence, some gonadal factors, probably the estrogen-PRL link, apparently amplify the LH effect on ß-GAL expression in adrenal glands. A similar finding was made in our previous study with the bLHß-CTP mice (8). After gonadectomy of LHR/ß-GAL mice, adrenal ß-GAL expression was strongest in zona reticularis, whereas in LHR(ß-GAL)/bLHß-CTP double-TG mice (with intact gonads), ß-GAL expression was confined mainly to zona glomerulosa and zona fasciculata. The shortest (173 bp) LHR promoter directed the ß-GAL expression to testes, but only the longest (7.4 kb) LHR promoter brought about adrenal expression. The 173-bp and 7.4-kb promoters were previously found also to direct differential splicing of ß-GAL mRNA in the testis (23). These findings support the observation that the alternative splicing of LHR transcripts is tissue specific and the quality of transcription is hormonally regulated (21, 22).

In the present study, we found that ß-GAL expression in the ovary and adrenal gland was activated by an identical LHR 5'-flanking region (7.4 kb), at least under conditions of elevated LH secretion. Conspicuously, no ß-GAL expression was found in granulosa and luteal cells, indicating that the regulation of LHR expression in these cells must be very different. The mode of ß-GAL expression in the ovary and adrenal gland differed from the testicular expression of intact LHR/ß-GAL TG mice. Only the longest LHR promoter analyzed (7.4 kb) directed expression of full-length ß-GAL to the adrenal gland of gonadectomized LHR/ß-GAL mice and the ovary and adrenal gland in the double-TG LHR(ß-GAL)/bLHß-CTP female mice. In contrast, the shortest promoter (173 bp) drove expression of the full-length ß-GAL transcript only to the testes of intact LHR/ß-GAL mice with normal LH levels. When LH levels rise, the intensity of ß-GAL expression increases in adrenal and ovary.

Several studies on transcription initiation sites of the LHR gene have been carried out, but the results are still conflicting (45, 46, 47, 48, 49). The inconsistent results may be due to the different species studied, cell lines used, and stage of the estrous cycle as well as to other hormonal conditions. An interesting finding of our previous study was that the shortest (173 bp) promoter directed mainly the full-length ß-GAL transcript expression, with transcription initiation site close to the ATG codon. In contrast, the longer promoters directed transcription mainly of truncated messages, with transcription initiation sites mainly localized higher upstream (23). The results indicate that the full-length and alternatively spliced transgenic mRNAs have specific transcription initiation sites and the up-stream promoter sequence, at least in the case of LHR, contributes to the alternative splicing. RT-PCR demonstrated that in the ovary and adrenal gland of LHR(ß-GAL)/bLHß-CTP mice with the 7.4-kb promoter, the 5'-ends of the transgene transcripts stretched upstream of bp -227. In contrast, the testes of intact LHR/ß-GAL mice revealed no transcripts with such long 5'-untranslated regions. 5'-RACE of the endogenous LHR message in the ovary, testis, and C{alpha}1 cells supported the RT-PCR results by demonstrating that the ovarian and adrenal tumor transcripts start farther upstream of those of the testis. This finding indicates that different promoter elements are used by the different tissues expressing the LHR gene. Moreover, the findings on transcription initiation sites confirm the contention that tissue-specific factors are involved in control of the LHR transcription and mRNA splicing.


   Acknowledgments
 
We thank Dr. John H. Nilson and Dr. Ruth A. Keri for allowing us to use the bLHß-CTP mice and for valuable comments on the manuscript. The skillful technical assistance of Ms. Nina Messner, Ms. Riikka Kytömaa, and Ms. Johanna Vesa is gratefully acknowledged. Transgenic mice were produced at the Transgenic Mouse Core Facility, University of Turku.


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

Abbreviations: ß-GAL, ß-Galactosidase; bLHß-CTP, bovine LH ß-subunit/C-terminal fragment of human chorionic gonadotropin-ß;C{alpha}1, adrenal tumor cell; LHR, LH receptor; PRL, prolactin; RACE, rapid amplification of cDNA ends; TG, transgenic.

Received February 11, 2002.

Accepted for publication June 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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