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Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland
Address all correspondence and requests for reprints to: Dr. Ilpo T. Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The putative promoter area in the 5'-flanking region of the LHR gene has been sequenced from the rat, mouse, and human, and certain species differences have been observed (7, 8, 9, 10, 11, 12, 13). Rat and mouse studies have demonstrated that the TATA-less promoter has the basal transcriptional activity within the first 173 bp upstream of the translation initiation codon (8, 9). The transcription initiation sites in the rat ovary have been localized at -13 bp (10) and -14, -19, -33, and -48 bp (11) upstream of the translation initiation codon. For mouse testicular LHR expression, the major transcription initiation site is at -310 bp (12). In contrast to those in the rat and mouse, the 5'-untranslated region (5'-UTR) of the human LHR gene contains two TATA boxes and a CAAT box, and its transcription initiation site in the human testis is localized as far as -1085 bp upstream of the translation initiation codon (7). Studies on a recently found new human LHR gene, apparently an allelic variant (14), isolated from the human placental genomic library, revealed transcription initiation sites in the ovary at positions -2, -6 -18, -37, and -70 bp (13). Hence, there are considerable species differences in the length of the 5'-UTR of the LHR gene, and some data allude to differences in ovarian and testicular expression within the same species.
The ontogeny of LHR expression is best known in the rat. In the male, it starts in Leydig cells around embryonic day 15 (E15) of the fetal period, declines transiently in early postnatal life along with the disappearance of fetal Leydig cells, and increases again at puberty and through adult life with the appearance of the adult population of Leydig cells (15). In the ovary, LHR is found in interstitial, thecal, and granulosa cells and in corpus luteum (16). In immature rat ovary, LHR is expressed in thecal cells, and after stimulation with PMSG, LHR messenger RNA (mRNA) is found in both granulosa and thecal cells (17). Truncated forms of LHR mRNA are expressed in the fetal ovary, but functional LHR does not appear until day 7 postnatally (18). A similar development pattern has been observed for LHR expression in the mouse (19).
The LHR gene expresses several mRNA splice variants in the testis and ovary (20, 21, 22). By a sensitive RT-PCR method, a transcript corresponding to the extracellular domain of the LHR can be first detected on E13.5 in rat testis and ovary (23). The full-length mRNA appears on E15.5 in the rat testis (24) but as late as postnatal day 7 in ovary (18, 25), and on day 5 in the mouse ovary (19). The appearance of the full-length LHR mRNA coincides with the onset of LHR binding (18, 26) and LH stimulated cAMP and testosterone production (26, 27).
Leydig cells exist in the testes as two growth phases. The fetal-type Leydig cells appear around E15 and persist until 2–3 weeks of postnatal life; testosterone production by them is responsible for male genital masculinization. The adult-type Leydig cells are responsible for sexual maturation at puberty (15) and testosterone supply for the adult male. The autologous regulation of LHR expression in the testis is dependent on the developmental stage of Leydig cells. Whereas LH/hCG clearly up-regulates LHR expression in the fetal-neonatal rat testes, similar stimulation down-regulates LHR expression in adult testes (28, 29). Hence, there are also clear-cut age-dependent differences in the regulation of LHR expression and function.
The purpose of this study was to examine more closely the tissue- and cell-specific function of the murine LHR promoter. Therefore, transgenic (TG) mice were created, expressing under a 2-kb fragment of the murine LHR promoter, the ß-galactosidase (ß-GAL) reporter gene. Surprisingly, the promoter fragment used appeared to be both age and sex specific in its function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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The studies were approved by the Turku University ethical committee for the care and use of experimental animals.
Northern hybridization analysis
Total RNA was isolated from tissues and cells using the single
step acid guanidinium thiocyanate-phenol-chloroform extraction method,
as described previously (33). 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 (RPN 303 N, 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 and the extracellular part of LHR, for
hybridization to transcripts of the transgene in tissues and of
endogenous LHR gene in cell cultures, respectively, were generated
using a Riboprobe System II kit (Promega Corp., Madison,
WI). Hybridizations and washings of the membranes were performed
according to the instructions of the membrane manufacturer.
Hybridization was detected by autoradiography using Kodak
film (XAR 5, Eastman Kodak Co., Rochester, NY).
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; fixed for 15–30 min, depending on the size of
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; 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 for 16 h 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] (34). After staining, the tissues were washed two or
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. Thereafter, the sections were photographed under microscope using a digital camera (DC 100, Leica Corp., Heerbrugg, Switzerland). The brain tissues were sectioned at the site of ß-GAL staining, and the photographs were taken from the tissue block under the microscope on Kodak 200 film (Eastman Kodak Co.), focused at the cut surface.
Immunohistochemistry
Fixed and ß-GAL-stained 5-µm paraffin sections of testis
were dewaxed and incubated in 1.5%
H2O2-absolute ethanol for 10 min during
rehydration. The sections were coated with 3% normal goat serum in
Tris-buffered saline for 30 min at room temperature. After washing, a
polyclonal rabbit antirat LHR antiserum (donated by Dr. H. Rajaniemi,
University of Oulu, Oulu, Finland), diluted 1:200 to 1:2000 in
Tris-buffered saline was added, and the slides were incubated at 4 C
overnight. The sections were washed and coated with the secondary
antibody, biotinylated antirabbit IgG (dilution, 1:250), and incubated
for 1 h at room temperature. For visualization of the bound
antibodies, the immunoperoxidase technique (Vectastain
Elite ABC kit, Vector Laboratories, Inc., Burlingame, CA)
was used according to instructions of the manufacturer. Counterstaining
was not used. The sections were dehydrated and photographed under the
microscope by digital camera (Leica DC 100).
RT-PCR
Five micrograms of total RNA were treated with deoxyribonuclease
I (Life Technologies, Inc., Paisley, Scotland, UK) and
reverse transcribed using the AMV reverse transcriptase (Promega Corp.) and a gene-specific antisense primer corresponding to
nucleotides 590–569 (5'-TCCAGATAACTGCCGTCACTC-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 -30–13 (5'-ACCGGAGCTCACACTCAG-3') of the
LHR 5'-flanking region. To discriminate between RNA and genomic DNA
contaminations, the primers were designed to flank the ß-globin
intron.
Rapid amplification of 5'-cDNA ends (5'-RACE) and Southern
hybridization
Five micrograms of total RNA from testis and brain tissues were
reverse transcribed using a 5'/3' RACE kit (Promega Corp.). A gene-specific antisense primer corresponding to
nucleotides 1278–1259 (5'-GGTCAGACGATTCATTGGCAC-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, a nested gene-specific
primer corresponding to nucleotides 590–569
(5'-TCCAGATAACTGCCGTCACTC-3'), and the oligo(deoxythymidine) anchor
primer were used to amplify the tailed cDNA in PCR. Fifteen microliters
of the PCR product were loaded on 1.5% agarose gel for electrophoresis
and transferred onto Hybond N nylon membrane by capillary blotting
overnight. For Southern hybridization, an oligo probe corresponding to
nucleotides 237–218 (5'-TCAGGAAGATCGCACTCCAG-3') of the ß-GAL-coding
sequence was end labeled with [
-32P]ATP
(Amersham Pharmacia Biotech). After hybridization, the
membrane was exposed to Kodak film (XAR 5).
Circular PCR and sequencing
The yield of the 5'-RACE products isolated from agarose gel was
too low for automated sequencing. To increase the concentration of the
DNA template for sequencing, circular PCR, modified by the method of
circular RACE-PCR described by Barth et al. (35), was used.
The 5'-RACE products isolated from agarose gel were circularized by
ligating the DNA using the SureClone Kit (Promega Corp.).
This DNA was subjected to circular PCR, designed so that the PCR
amplification reaction crossed the ligation boundary and the 5'-end of
the transcripts using two primers in reverse orientation. The PCR
products were isolated from agarose gel and subjected to automated
sequencing (ABI Prism 377 DNA Sequencer, Perkin-Elmer Corp., Foster City, CA).
Cell culture transfections
Murine Leydig tumor cells, mLTC-1 (36), were cultured in
buffered Waymouth’s medium (Life Technologies, Inc.) with
9% horse serum, 4.5% FCS, and 50 mg/liter gentamicin. Murine
granulosa tumor cells, KK-1 (37), and human cervical carcinoma cells,
HeLa, were cultured in DMEM-M12 medium (1:1; Life Technologies, Inc.) with 10% FCS and 50 mg/liter gentamicin.
Six million cells were transiently cotransfected with 30 µg of the LHR/ß-GAL construct and 3 µg of pCMV-luciferase as a control of transfection efficiency, using the electroporation method (330 V, 960 µF; Bio-Rad Laboratories, Inc., Richmond, CA). The transfected cells were cultured for 24–36 h, harvested, and lysed. The lysates were examined for ß-GAL and luciferase activities using light spectrophotometer and 1251 luminometer (BioOrbit, Turku, Finland), as described by Sambrook et al. (38). Luciferase activity was used for correction of the transfection efficiency of the cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern hybridization analysis showed that besides the full-length
ß-GAL transcript (3.2 kb), shorter forms were also expressed in the
testis. A truncated 2.3-kb mRNA was quantitatively most abundant, but
we have not identified which part of the ß-GAL is spliced out, and
whether this mRNA is translated into functional enzyme. No ß-GAL
transcripts were detected in the ovary or in control RNA isolated from
liver and kidney (Fig. 2). Moreover, as
demonstrated in Fig. 2, considerable differences were observed in the
levels of transgene expression between the different lines.
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). The patchy staining
pattern indicates that high ß-GAL expression was not present (or of
similar intensity) in all Leydig cells. By immunohistochemistry we
confirmed that the transgene ß-GAL and endogenic LHR were expressed
in same cells (Fig. 3, c and d). The expression of ß-GAL is clearly
seen in Leydig cell cytoplasm and that of endogenous LHR on cell
membrane of the same cells. The testicular expression of ß-GAL was
age dependent, and it was found from week 5 onward in the Leydig cells.
The expression in elongating spermatids was found as soon as this cell
type appeared in the developing testis, i.e. from week 4
onward. In the neonatal testis, ß-GAL expression was totally absent
in Leydig cells, but was present in some spermatogonia, and between the
ages of 1–3 weeks, testicular expression was totally absent (Fig. 4). The expression reappeared at the age
of 4 weeks to elongating spermatids and 1 week later to Leydig cells
and persisted thereafter throughout adult life. Histochemical studies
of ovaries revealed weak ß-GAL expression in granulosa cells, but
staining of similar intensity was present in TG and non-TG mice,
surrounding some of the developing follicles (results not shown).
Hence, all of the ovarian staining was considered to represent
endogenous ß-GAL expression.
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). In addition, a few pituitary samples
displayed weak ß-GAL staining (results not shown). In particular, the
following extragonadal tissues, shown by others to express the LHR mRNA
(1, 3, 6, 39), were found to lack transgene expression: uterus, adrenal
gland, and seminal vesicles.
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). The expected 700-bp full-length
message of the transgene, after splicing out the ß-globin intron, was
present in both tissues, but was less abundant than the truncated
forms. This is apparently to some extent due to more effective
amplification of the shorter cDNA forms in PCR, which thus
overestimates their relative quantities. However, as the Northern
hybridization demonstrates (Fig. 2
), truncated splice variants of the
transgene dominate in the testis. When ovarian RNA was used as
template, no detectable PCR products were found, nor did the RNAs from
muscle, spleen, kidney, and adrenal gland yield amplification products
(Fig. 6). 5'-RACE produced three transcripts from testicular RNA and
two from brain RNA (Fig. 7). Two of these
products (250 and 350 bp) were of equal size in testis and brain, the
third one (700 bp) was longer in the testis. Subsequent circular PCR
revealed two main amplification products in the testis, and sequencing
of these products located the transcription initiation around
nucleotide -20 of the LHR 5'-flanking region. The circular PCR
products from brain samples were not sequenced.
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). Moreover, no ß-GAL
expression was found in the nongonadal HeLa cells that were used as a
negative control. The endogenous LHR expression was confirmed by
Northern hybridization of total RNA of mLTC-1 and KK-1 cells (Fig. 8b)
The expression patterns of LHR mRNA splice variants in testicular and
ovarian cells were according to previous findings (24, 25, 40),
i.e. a 1.8-kb form dominating in Leydig cells and a 4.2-kb
form dominating in granulosa cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that the transgene was expressed, besides the expected full-length mRNA, also in several truncated splice variants appears to be a common finding with transgenes (41, 42). Because of the same tissue distribution of the splice variants and the presence of functional ß-GAL protein, the aberrant mRNA does not affect the interpretation of the data, which provides qualitative information on tissue distribution of the transgene expression.
The expression of the LHR/ß-GAL fusion gene in some spermatogonia of
neonatal testes and in elongating spermatids of the pubertal and adult
testis, was an unexpected finding. The seminiferous epithelial
expression level seemed to parallel the serum testosterone level or the
presence of functional Leydig cells in the testis; it was detectable in
neonatal spermatogonia, absent between 1–3 weeks of age, and
reappeared at the age of 4 weeks. No reports exist on expression of the
endogenous LHR gene in spermatids, and this is not apparent in in
situ hybridization data of LHR mRNA in the testis (40). Whether
this represents aberrant expression of the transgene or is indicative
of endogenous LHR expression remains to be studied. It should be stated
that we previously observed seminiferous tubular expression of a FSH
ß-subunit promoter-driven transgene (41), and subsequent studies
showed that the endogenous common 
-subunit, LHß, and FSHß genes
are also expressed at low level in spermatogenic cells (43, 44). Hence,
the elements of gonadotropin-driven regulatory system are expressed
within the seminiferous tubules.
The fact that the 2-kb promoter of the LHR gene was unable to express LHR in fetal Leydig cells emphasizes further the special functional features of this cell type. We have previously shown that fetal Leydig cells are resistant to the attenuating effects of high LH/hCG stimulation on LHR expression and androgen biosynthesis (28). Instead, high gonadotropic stimulation increases LHR expression and androgen synthesis (29). The present findings are therefore in good agreement with the functional data showing that the regulation of LHR expression is different in fetal and adult Leydig cells. Moreover, the patchy staining pattern of the adult interstitial space indicates that high ß-GAL expression was not present in all Leydig cells. The 2-kb LHR promoter may thus be functionally competent only at a special functional stage of the adult Leydig cells.
There are also other data on qualitative differences in LHR expression in different functional stages of Leydig cells. The studies with EDS-treated rats with selectively destroyed Leydig cells indicate that Leydig cell precursors express a truncated form (1.8 kb) of LHR mRNA and that the beginning of the process when Leydig cell precursors start to differentiate into mature Leydig cells occurs in the absence of LH, whereas LH and the expression of full-length LHR message are essential for complete differentiation of Leydig cells (45, 46, 47).
Another interesting finding of the present study was that the LHR/ß-GAL gene construct was not competent for expression in ovarian cells. Some ovary-specific cis-regulatory elements are absent in the 2-kb promoter sequence. Identification of these sequences will be of special interest. Likewise, the trans-activating elements needed for ovarian and testicular expression of the LHR gene need to be characterized. To this end, we are currently testing longer 5'-flanking sequences of the LHR gene in cell lines and new transgenic mice.
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Acknowledgments |
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Footnotes |
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Received June 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit promoter/simian virus T-antigen fusion
gene: characterization of ovarian tumors and establishment of
gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616–627-subunit are expressed in mouse testis, as determined in wild
type mice and those transgenic for the FSH ß-subunit/herpes simplex
virus thymidine kinase fusion gene. Endocrinology 136:4769–4775[Abstract]
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