Expression of the Mature Luteinizing Hormone Receptor in Rodent Urogenital and Adrenal Tissues Is Developmentally Regulated at a Posttransla
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内分泌学杂志 2005年第8期
Biocenter Oulu and Departments of Anatomy and Cell Biology (P.M.A., U.E.P.-R.) and Anatomy and Cell Biology (J.T.A., H.J.R.), University of Oulu, FI-90014 Oulu, Finland
Address all correspondence and requests for reprints to: Dr. Ulla Pet?j?-Repo or Dr. Hannu Rajaniemi, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, University of Oulu, FI-90014 Oulu, Finland. E-mail: ulla.petaja-repo@oulu.fi or hannu.rajaniemi@oulu.fi.
Abstract
The LH receptor (LHR) is a G protein-coupled receptor involved in the regulation of ovarian and testicular functions. In this study we demonstrate novel and unexpected patterns of receptor expression and regulation in fetal and adult rodent urogenital and adrenal tissues. Two rat LHR promoter fragments (2 and 4 kb) were shown to direct expression of the lacZ reporter in transgenic mice to gonads, adrenal glands, and kidneys, starting at 14.5 d post coitum, and to genital tubercles, starting at 11.5 d post coitum. These tissues were also found to express the full-length LHR mRNA and protein during rat fetal development, but, importantly, only immature receptors carrying unprocessed N-linked glycans were detected. After birth, the receptor gene activity ceased, except in the gonads, which started to express the mature receptor carrying fully processed N-linked glycans. Surprisingly, both LHR mRNA and mature protein levels were up-regulated substantially in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital tissues. Taken together, these results indicate that the LHR protein is expressed constitutively in gonadal and nongonadal urogenital tissues as well in adrenal glands, but its final functional maturation at the posttranslational level appears to be developmentally and physiologically regulated.
Introduction
THE LH RECEPTOR (LHR), like other glycoprotein hormone receptors, the FSH and TSH receptors, belongs to the leucine-rich repeat containing G protein-coupled receptors (GPCRs), with a large extracellular N-terminal domain that is involved in ligand binding (1). This integral membrane protein plays an essential role in the regulation of reproductive functions and is expressed in ovarian granulosa, thecal, interstitial, and luteal cells and in testicular Leydig cells (1). However, it appears to have a wider expression than was previously believed and has also been found to be expressed elsewhere in the reproductive tract (2, 3, 4) and in the placenta (2), umbilical cord (5), prostate (6), adrenal gland (7, 8, 9), and nervous tissue (3, 10, 11). Neither the functional role nor the regulation of nongonadal LHR expression is known at present.
Previous studies have shown that a substantial portion of LHRs in the gonads and nervous tissues as well as in heterologous expression systems exists in an immature form (11, 12, 13, 14), containing high mannose-type N-linked glycans, which are typical for proteins located in the endoplasmic reticulum (ER) (15). This suggests that they might mature inefficiently, a phenomenon that has been found to characterize a few other GPCRs (16, 17, 18, 19, 20). This idea is supported by our recent finding that in stably transfected human embryonic kidney 293 cells, about 80% of newly synthesized rat LHRs is retained in the ER and is targeted for ER-associated degradation (51). Because blockade of the degradation pathway was found to lead to enhanced ER export and maturation of the receptors, LHR expression at the cell surface may be controlled at the ER level. To determine whether maturation of LHRs could be a regulated phenomenon in vivo, we set out to examine the distribution and ontogeny of receptor expression in fetal and adult rodents in more detail, giving special attention to the developing urogenital structures (the term urogenital structures includes gonads, kidneys, genital tubercles, and adrenal glands). To this end, rat LHR promoter-driven transgene activity was assessed in transgenic mice, and receptor expression in fetal and adult rats was determined at the mRNA and protein levels. We demonstrate that during fetal development the receptor is expressed in various urogenital structures: the developing gonads, kidneys, genital tubercles, as well as adrenal glands. However, only immature receptors were detected; mature receptors were found only in adult gonads and, surprisingly, in pregnant female adrenal glands and kidneys. These results thus indicate that the abilities of the different tissues to synthesize and process the LHR protein may be developmentally and physiologically regulated.
Materials and Methods
Promoter constructs and production and analysis of transgenic mice
The construction, production, and analysis of transgenic mice were previously described (11). The rat LHR promoter-driven transgene constructs carried –2060 or –3970 bp from the translation initiation site of the rat LHR gene in front of the reporter gene lacZ. Pregnant females, neonates, and adult mice (60 d) were killed by inhalation of carbon dioxide, and fetuses and tissue samples from adult mice were collected, prefixed, and stained for ?-galactosidase (?-Gal) activity using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (11). After postfixation, the fetuses were observed as whole mounts under a stereomicroscope or, alternatively, were dissected for isolation of urogenital tissues. The paraffin-embedded tissue sections (10 μm) were counterstained with safranin.
Rat tissue preparation
Sprague Dawley rats were used for collecting tissues for RT-PCR and receptor purification. Pregnant females, neonates, and adult rats (90 d old) were killed, and tissue samples were removed, frozen in liquid nitrogen, and stored at –80 C. Dissection of fetal tissues was performed under a stereomicroscope. The use of animals was approved by the University of Oulu committee for the care of experimental animals.
RT-PCR
Four oligonucleotide primers of 29–36 nucleotides were designed based on the rat LHR sequence (GenBank accession no. M26199) (21), as previously described (11). Primers 1 (nucleotides –27 to +9 from translation initiation site) and 2 (nucleotides +2116 to +2084) were used for the RT-PCR, and primers 3 (nucleotides +65 to +97) and 4 (nucleotides +1887 to +1858) were used for the subsequent nested PCR. The expected molecular size of the nested PCR-amplified cDNA product was 1.9 kb, slightly smaller than the original full-length cDNA of 2.1 kb.
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA), according to the manufacturer’s instructions. The RT-PCR was carried out as described previously (11) in a total volume of 50 μl, using 1 μg total RNA as template and 0.2 μM primers 1 and 2. For the second amplification round, 1 μl of the product from the first amplification was used as a template with the slightly intended primers 3 and 4 in the same PCR conditions as in the first round. The PCR products (20 μl) were analyzed on a 1% agarose gel containing ethidium bromide.
Immunoprecipitation, ligand affinity chromatography, and Western blotting
Total cellular membranes were prepared by homogenizing with a Polytron homogenizer (Ultra-Turrax T-25, Janke & Kunkel, Staufen, Germany) in buffer A (PBS, pH 7.5, containing 5 mM EDTA, 5 mM N-ethylmaleimide, and 0.2 mM phenylmethylsulfonylfluoride; 100 mg/ml) and centrifuging at 100 x g for 10 min. The membrane particles were then collected by centrifuging the supernatant for 30 min at 45,000 x g. After washing with buffer A, the protein concentration was determined by Bio-Rad’s DC assay kit, using BSA as the standard. Tissue samples (10–60 mg/sample) from 20–30 fetuses [15.5–18.5 d post coitum (dpc)] and four to eight 1-d-old female or male neonates were pooled for membrane preparation. Membranes from the fetuses, neonates, and adult male testes (8 mg) and 15.5 and 19.5 dpc pregnant (1 mg) and nonpregnant (0.5 mg) female ovaries, adrenal glands (1 mg), and kidneys (6 mg) were then solubilized by stirring on ice for 60 min in 500 μl buffer B [buffer A containing 0.5% (wt/vol) Triton X-100 and 20% (vol/vol) glycerol] and centrifuging at 100,000 x g for 60 min. The soluble fraction was supplemented with 0.1% (wt/vol) BSA and subjected to immunoprecipitation or human chorionic gonadotropin (hCG) affinity chromatography, and the purified samples were analyzed by Western blotting, as described previously (11).
Results
LHR promoter-driven transgene expression in urogenital structures of developing and adult mice
To demonstrate LHR expression in the fetal and adult rodent urogenital structures, we first assessed LHR promoter activity in transgenic mice, which carry the Escherichia coli lacZ gene under control of the rat LHR promoter fragments of 2060 and 3970 bp (11). Both promoter fragments directed expression of the LacZ gene to the urogenital structures in a similar manner, and thus, a representative mouse line expressing the lacZ gene under the 3970-bp LHR promoter was chosen for additional analyses.
Expression of the lacZ gene was examined by whole mount staining of the fetal urogenital blocks or adult tissues with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside. Paraffin-embedded tissue sections (10 μm) were also examined to assess the transgene expression at the cellular level. As shown in Fig. 1A, ?-Gal activity was first detectable in the developing gonads in the 14.5-dpc fetuses, whereas no activity was apparent in the gonads of nontransgenic littermates (Fig. 1B). In the developing ovaries, ?-Gal activity remained at a low level (Figs. 1C and 2, A and B) until in the nearly fertile 30-d-old mice, in which distinct staining in the developing follicles was detected (Fig. 2C). In the adult females, transgene activity was clearly apparent in the maturing ovarian follicles and corpora lutea (Fig. 2D) and was located in granulosa, thecal, and luteal cells and in a varying degree to the ova and interstitial glandular cells (Fig. 2M). The corresponding nontransgenic littermates showed no staining (Figs. 1D and 2, E and F). Some specific transgene activity was also detected in the adult female uterus, but was not studied further. In the developing testes, specific ?-Gal expression was detected in the interstitial tissue, but not in the tubules (Figs. 1, E and F, and 2, G–L), and was clearly restricted to Leydig cells in adult testes (Fig. 2N). ?-Gal activity in the epididymis and ductus deferens was due to endogenous enzyme activity (Fig. 2, compare G to K and J to L), and expression of the transgene in these tissues could therefore not be examined.
FIG. 1. Expression of the LHR promoter-driven transgene in pre- and neonatal mouse urogenital structures. Dissected urogenital blocks of transgenic fetuses at age 14.5 dpc (A) and 16.5 dpc (C and E), and adrenal glands from 1-d-old neonates (G) were analyzed in situ for ?-Gal activity of the rat LHR promoter-LacZ construct, as described in Materials and Methods. The corresponding nontransgenic samples are presented in B, D, F, and H. In the 14.5-dpc mice (A), expression was detected in developing gonads, adrenal glands, and kidneys. The same expression pattern was detected in the corresponding structures of the 16.5-dpc transgenic females (C) and males (E), but in a more dispersed manner. Adrenal glands of 1-d-old neonates (G) showed distinct staining. Ad, Adrenal gland; Go, gonad; Ki, kidney; Ov, ovary; Sg, sensory ganglion; Te, testis; Tg, transgenic; Wt, wild type.
FIG. 2. Expression of the LHR promoter-driven transgene in postnatal mouse gonads. Dissected gonads were stained as described in Fig. 1. The dispersed staining observed in the ovaries of transgenic 1-d-old (A) and 15-d-old (B) females became more intense in those of 30-d-old (C) and adult (D) females, in which ?-Gal activity was clearly apparent in developing follicles and corpora lutea (active and inactive corpora lutea are shown by arrowhead and arrow, respectively). Distinct staining in the testicular interstitial tissue could be seen in transgenic 1-d-old (G), 15-d-old (H), 30-d-old (I), and adult (J) males. Ovaries of nontransgenic 1-d-old (E) and adult female (F) mice showed no expression. The same was true for the testes of nontransgenic males (K and L), but the epididymis displayed endogenous ?-Gal activity. The paraffin-embedded transgenic adult ovaries (M) and testes (N) were sectioned (10 μm) and counterstained with safranin, as described in Materials and Methods. ?-Gal staining could be detected in ovarian interstitial tissue, in thecal and granulosa cells (arrow) of the developing follicles (M), and in testicular Leydig cells (arrow; N). Scale bars, 120 μm (M) and 20 μm (N). Ep, Epididymis; Se, seminiferous tubule; Tg, transgenic; Wt, wild type.
In the 14.5-dpc transgenic fetuses (Fig. 1A), ?-Gal activity could be detected not only in the gonads, but, surprisingly, also in the developing adrenal glands and kidneys. Branched tree-like expression in the kidneys was localized to the tubular epithelium, whereas in the adrenal glands, enzyme activity was detected in mesenchymal cells. Similar staining was detected in 16.5-dpc female (Fig. 1C) and male (Fig. 1E) fetuses. In 1-d-old neonates, distinct staining was found in the medulla and cortex of transgenic (Fig. 1G), but not nontransgenic (Fig. 1H), adrenal glands, after which it became undetectable. The medullary staining is most likely due to transgene expression in the ganglion cells that are of the same origin as those in sympathetic ganglia (11). In kidneys, endogenous ?-Gal activity was apparent in 1-d-old neonates, and the transgene and endogenous expression could therefore not be separated after birth.
Whole mount staining of the transgenic mouse fetuses revealed that the LHR promoter-driven transgene was also expressed in genital tubercles and paired genital swellings, representing the developing external genitalia. The expression was apparent in genital tubercles in the 11.5-dpc fetuses (Fig. 3A) and from 14.5 dpc onward also in the urogenital fold (Fig. 3C, arrow). At the cellular level, expression was localized to the outmost mesenchyme of the genital tubercle (Fig. 3K). In the 1-d-old neonates, ?-Gal activity was clearly apparent in the external genitalia in both females and males (Fig. 3, G and I, respectively), and no distinct sex-dependent differences were observed, although there was a clear difference in the anatomical shape of the corresponding structures. The nontransgenic littermates showed no ?-Gal staining (Fig. 3, D, F, H, J, and L).
FIG. 3. Expression of the LHR promoter-driven transgene in pre- and neonatal mouse genital tubercles. Dissected genital tubercles of transgenic fetuses aged 11.5 dpc (A), 13.5 dpc (B), 14.5 dpc (C), and 16.5 dpc (E) and from a 1-d-old neonatal female (G) and male (I) were stained as described in Fig. 1. The nontransgenic genital tubercles of 14.5- and 16.5-dpc fetuses and 1-d-old neonatal females and males are presented in D, F, H, and J, respectively. The arrows in C and E indicates the urogenital fold, and the arrows in G and H show the paired genital tubercle. The paraffin-embedded 14.5-dpc fetal transgenic (K) and nontransgenic (L) genital tubercles were sectioned (10 μm) and counterstained with safranin. Distinct ?-Gal staining could be detected in the outermost mesenchymal cell layer in the transgenic, but not nontransgenic, genital tubercles. Scale bars, 100 μm (K and L). Tg, Transgenic; Wt, wild type.
Taken together, these results indicate that during organogenesis the LHR promoter is first activated in the developing genital tubercles around 11.5 dpc and in the gonads, adrenal glands, and kidneys at around 14.5 dpc. After birth, activity decreases in adrenal glands and kidneys, but increases substantially in gonads at puberty, residing in specific cell types in mature ovaries and testes.
LHR mRNA expression in urogenital structures of developing and adult rats
To examine LHR expression in the developing urogenital structures, total RNA was isolated from fetal and adult rat tissues and subjected to RT-PCR, followed by nested PCR for amplification of the products. The primers used were selected to encode the entire coding region of the LHR (11). As expected, two distinct amplified products of 2.1 and 1.8 kb were detected after the first amplification round in adult testes and ovaries (Fig. 4A, upper panel, lanes 3 and 6, respectively), corresponding to the full-length LHR and the most common receptor variant, respectively (22, 23, 24). Occasionally, a third transcript of 2.0 kb was detected, most likely representing a hybrid of the 1.8- and 2.1-kb forms (3). In the second amplification round, the final products were slightly smaller than the original cDNA due to the nested primers used (Fig. 4A, lower panel, lanes 3 and 6). As shown in lanes 1 and 4 in Fig. 4A, the full-length transcripts were present in the testes as well as in the ovaries of 17.5-dpc fetuses, the youngest age studied, as well as in 1-d-old neonates (lanes 2 and 5). In developing testes, the transcripts were apparent after the first amplification round, but required additional amplification in the ovaries. The two major mRNA products were also obtained from fetal and neonatal adrenal glands (Fig. 5A, lanes 1–4) and kidneys (Fig. 5A, lanes 7–10) as well as from genital tubercles (data not shown). However, as in fetal and neonatal ovaries, the LHR mRNA transcripts were detected only after the second amplification round. When RNA from adult female adrenal glands was used as a template, the transcripts were detectable after the first amplification round (Fig. 5A, lane 5), whereas the male samples revealed transcripts only after the second round (Fig. 5A, lane 6). The adult kidneys yielded no amplification products (Fig. 5A, lanes 11 and 12). Taken together, these results suggest that the developing urogenital structures start to express full-length LHR mRNA early during differentiation. Although there seem to be no differences in the onset of mRNA expression in males and females, the amount of transcripts appears be higher in males, with the exception of the adrenal glands.
FIG. 4. Expression of LHR mRNA and protein in rat testes and ovaries. A, RT-PCR analyses of the total RNA from testes (lanes 1–3) and ovaries (lanes 4–7). The analyses were performed with primers flanking the entire coding region of the LHR to amplify the full-length cDNAs (upper panel), followed by nested PCR with the intended primers (lower panel), as described in Materials and Methods. Ovarian RNA without RT reaction (lane 7) was used as a negative reaction control. Lanes 1 and 4, 17.5 dpc; lanes 2 and 5, 1-d-old; lanes 3, 6, and 7, adult. The major amplified mRNA products are indicated on the left. B, Western blots of purified receptors from testes (lanes 1–3, 7, and 8) and ovaries (lanes 4–6, 9, and 10). Solubilized cellular membranes from the 15.5- to 18.5-dpc (lanes 1 and 4), 1-d-old (lanes 2, 5, 7, and 9), and adult (lanes 3, 6, 8, and 10) rat gonads were subjected to immunoprecipitation or ligand affinity chromatography and analyzed by Western blotting using a polyclonal antibody directed against the C-terminus of the receptor, as described in Materials and Methods. The mature receptor forms are indicated by arrows, and the immature ones are shown by arrowheads. *, Ig heavy chains. Mr markers are indicated on the right and left.
FIG. 5. Expression of LHR mRNA and protein in rat adrenal glands and kidneys. A, RT-PCR analyses of the total RNA from adrenal glands (lanes 1–6) and kidneys (lanes 7–12). RT-PCR and the subsequent nested PCR were performed as described in Materials and Methods. Lanes 1 and 7, 16.5-dpc female; lanes 2 and 8, 16.5-dpc male; lanes 3 and 9, 1-d-old female; lanes 4 and 10, 1-d-old male; lanes 5 and 11, adult female; lanes 6 and 12, adult male. B, Western blots of purified receptors from adrenal glands. Solubilized cellular membranes from the adrenal glands of 15.5- to 18.5-dpc fetuses (lane 1), 1-d old females (lanes 2 and 6), 1-d-old males (lanes 3 and 7), adult females (lanes 4 and 8), and adult males (lanes 5 and 9) were subjected to immunoprecipitation or ligand affinity chromatography and analyzed by Western blotting, as described in Materials and Methods. *, Ig heavy chains.
LHR protein expression in urogenital structures of developing and adult rats
To determine whether the full-length receptor mRNA detected in the developing urogenital structures is translated to protein, Triton X-100-solubilized membranes were subjected to immunoprecipitation using an LHR-specific polyclonal antibody directed against the C-terminal domain of the protein (25). The immunoprecipitated samples were then analyzed by Western blotting using the same antibody for detection. As we have previously documented (11), two receptor species with apparent molecular masses (Mr) of 90,000 and 73,000 were detected from the adult rat ovary (Fig. 4B, lane 6). These species were shown to carry complex-type and high-mannose-type N-linked oligosaccharides, respectively (11), thus representing mature and immature forms of the receptor. Two receptor species were also immunoprecipitated from the adult testes (Fig. 4B, lane 3), but the larger species migrated more slowly (Mr, 100,000), most likely due to tissue-specific differences in the glycan moieties. Importantly, both the mature and immature receptor species were purified by ligand (hCG) affinity chromatography (Fig. 4B, lanes 8 and 10), showing that they were able to bind hormone.
Surprisingly, none of the developing or adult nongonadal urogenital tissues were found to express the mature LHR, except for adult female adrenal glands, in which the mature receptor was just barely detectable (Fig. 5B, lanes 4 and 8). Only the 73,000 Mr receptor species was immunoprecipitated from fetal gonads (Fig. 4B, lanes 1 and 4), adrenal glands (Fig. 5B, lane 1), and kidneys (data not shown) as well as from 1-d-old female ovaries (Fig. 4B, lane 5), adrenal glands (Fig. 5B, lane 2), and kidneys (data not shown), whereas two receptor species of 73,000 and 70,000 Mr were purified from the corresponding 1-d-old male tissues (testes: Fig. 4B, lanes 2; adrenal gland: Fig. 5B, lane 3). These two receptor species were also purified from female and male fetal and neonatal genital tubercles (data not shown). Both the 73,000 and 70,000 Mr receptor forms were also detected by a polyclonal antibody that was raised against the N-terminal domain of the rat LHR (25), ruling out the possibility that the 70,000 Mr species might represent a degradation product of the larger one.
In summary, these results demonstrate that a mature LHR species carrying fully processed N-linked oligosaccharides appears to be expressed only in the gonads of adult rats and to some extent in the adult female adrenal glands, whereas only immature receptor forms are expressed in fetal and neonatal rat urogenital structures. This apparent tissue- and age-dependent difference in the ability of cells to process the LHR protein to the mature form may thus be developmentally and physiologically regulated.
LHR expression in adrenal glands and kidneys of pregnant rats
The results presented above suggest that the LHR is expressed in fetal adrenal glands and kidneys and to some extent also in adult female adrenal glands. This finding together with the previous observation that receptor expression in adrenal glands may be hormonally regulated (8, 26) prompted us to determine whether LHR expression might be up-regulated during pregnancy. To this end, tissue samples from pregnant female rats of 15.5 and 19.5 dpc were isolated and analyzed. As expected, the mRNA expression level in the ovaries was higher in pregnant females compared with nonpregnant animals (compare Fig. 6A, lanes 1 and 2, to Fig. 4A, lane 6). Importantly, mRNA expression was also substantially higher in the adrenal glands and kidneys. The transcripts were clearly detectable at 15.5 dpc after the first amplification round (Fig. 6A, lanes 3 and 5, respectively), which was in clear contrast with the findings obtained for nonpregnant females (see Fig. 5A, lanes 5 and 11). However, unlike in the ovaries, expression decreased at 19.5 dpc (Fig. 6A, lanes 4 and 6) and was detected in the kidneys only after the second amplification round (data not shown).
FIG. 6. Expression of LHR mRNA and protein in ovaries, kidneys, and adrenal glands of pregnant female rats. A, RT-PCR analyses of the total RNA from ovaries (lanes 1 and 2), adrenal glands (lanes 3 and 4), and kidneys (lanes 5 and 6). The analyses were performed as described in Materials and Methods. Lanes 1, 3, and 5, 15.5-dpc pregnant females; lanes 2, 4, and 6, 19.5-dpc pregnant females. B, Western blots of immunoprecipitated (lanes 1–3) or ligand affinity purified (lanes 4–6) receptors from pregnant (15.5 dpc) female ovaries (lanes 1 and 4), adrenal glands (lanes 2 and 5), and kidneys (lanes 3 and 6). The samples were analyzed as described in Materials and Methods. *, Ig heavy chains.
To determine whether the high amount of mRNA transcripts detected in the adrenal glands and kidneys during pregnancy might correlate to the expression of mature receptors in these tissues, samples were prepared for immunoprecipitation. As shown in Fig. 6B, the LHR-specific antibody recognized two receptor species, of 90,000 and 73,000 Mr, from the ovaries (lane 1) as well as the adrenal glands (lane 2) and kidneys (lane 3) of 15.5-dpc pregnant females. Both of these receptor species were purified by ligand affinity chromatography (Fig. 6B, lanes 4–6), indicating that they are capable of hormone binding. Near the time of delivery, at 19.5 dpc, the expression decreased to approximately the same level as in nonpregnant females, and the adrenal glands and kidneys again appeared to contain mostly the immature LHR form (data not shown). Only the adrenal gland displayed a small amount of receptor corresponding to the mature form.
Thus, these results demonstrate that although the immature LHRs are the predominant receptor species detected in adult female rat adrenal glands, there is a clear shift to an increased level of mature receptors during pregnancy in both adrenal glands and kidneys. This finding suggests that LHR expression may be hormonally regulated in these nongonadal tissues.
Discussion
In the present study we demonstrate that the LHR is expressed not solely in the gonads, but is also present in other urogenital structures, such as kidneys and genital tubercles as well as adrenal glands. The receptor gene activity appears to start early during fetal development, but importantly, the receptor protein is expressed only in an immature form. Mature receptors were detected only in adult gonads and, surprisingly, in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital structures. These results implicate new and complex roles for LH and CG and indicate that the expression of mature and functional LHRs is developmentally and physiologically regulated in both gonadal and nongonadal urogenital tissues.
The tissue- and age-dependent expression of the LHR in urogenital structures was first examined using transgenic mice carrying the lacZ reporter under the rat LHR promoter. The two promoter fragments of 2060 and 3970 bp used directed ?-Gal expression in a similar manner to developing and adult tissues, and as expected, the reporter activity was detected in specific gonadal cells in adult mice, testicular Leydig cells, and ovarian thecal, granulosa, and luteal cells. In the developing fetuses, reporter gene activity was first detected in the gonads at 14.5 dpc, concomitantly with the adrenal glands and kidneys, whereas the genital tubercles showed activity even earlier at 11.5 dpc. In contrast to these findings, H?m?l?inen and co-workers (9, 27, 28) were unable to detect transgene expression in prenatal transgenic mice when mouse LHR promoter fragments of 7.4, 2.1, or 173 bp were used. Furthermore, the mouse promoter fragments were found to show clear sex- and tissue-dependent differences in activity in adult tissues. The reasons for these differences are not known, but they may be due to species differences or differences in the length and usage of the 5'-flanking sequences of the two genes.
The findings obtained with transgenic mice were substantiated by demonstrating LHR expression in various rat urogenital structures at the mRNA level. Two distinct mRNA species of 2.1 and 1.8 kb, corresponding to the full-length LHR and the most common variant, respectively (22, 23, 24), were amplified from fetal and adult rat tissues; importantly, no apparent sex differences in the onset of mRNA expression were observed. For example, the full-length mRNA transcripts were detected in the gonads at 17.5 dpc in both female and male fetuses. This is in contrast with previous findings that rat ovaries were found to express only truncated mRNA species before birth (29). These apparent differences are probably due to the low amount of full-length receptor transcripts in the developing ovaries that may have hindered their detection previously, especially because different techniques to detect mRNA expression were used. In the present study the transcripts were detectable in fetal ovaries by RT-PCR only after the second amplification round, whereas those in the testes were easily detectable after the first round. This sex-dependent difference in the amount of receptor transcripts in the fetal gonads was not unexpected, however, because the same difference was clearly apparent in adult gonads as well.
The LHR expression in developing and adult urogenital tissues was also demonstrated at the protein level. Immunoprecipitation using a receptor-specific antibody revealed two receptor species of 90,000 and 73,000 Mr from adult rat ovaries and pregnant rat adrenal glands and kidneys, whereas 100,000 and 73,000 Mr species were obtained from adult testes. Similar receptor species have been found previously in adult rat and porcine gonads (12, 13, 30, 31, 32, 33, 34, 35) and in fetal and adult rat nervous tissue (11) as well as in various heterologous expression systems (12, 14, 51). The 90,000 and 100,000 Mr receptor forms correspond to fully mature receptors, because they have been shown to contain complex type N-linked oligosaccharides, and their size difference is due to tissue-specific glycosylation (11, 12, 13, 14, 33, 36). The 73,000 Mr species, in contrast, is likely to represent immature receptor precursors, because it carries high-mannose-type N-linked glycans (11, 12, 13, 14, 37), which are typical for glycoproteins residing in the ER. Unlike in the adults, fetal urogenital tissues were found to express not mature receptors, but only two smaller species of 73,000 and 70,000 Mr. Because both of these forms were recognized by antibodies directed against the N- and C-terminal domains of the receptor, the smaller species does not represent a degradation product of the larger one, but, instead, may be an intact receptor precursor with a fewer number of N-linked glycans than the 73,000 Mr form. The scarcity of the tissue samples prevented us from testing this possibility in more detail. Alternatively, the 70,000 Mr receptor form might result from alternative splicing of the primary transcript and represent a receptor isoform lacking some of the exons coding for the extracellular domain of the receptor (22, 23, 24). This is an interesting possibility, because alternatively spliced transcripts are especially abundant in male gonads (38, 39), in which the 70,000 Mr receptor species was the most abundant one.
It has been suggested that LHR expression in the developing gonads is regulated at a posttranscriptional level, because the onset of expression of receptors capable of hormone binding in the developing testes and ovaries has been reported to coincide with the change in the alternative splicing pattern of the LHR mRNA (29, 39). Our results extend these findings and argue that the expression of fully functional mature receptors is also regulated at a posttranslational level in both gonads and nongonadal urogenital tissues. This idea was supported by the fact that there was a clear age-, tissue-, and sex-dependent variation in the relative amount of mature receptors to immature ones. Furthermore, the expression of mature receptors was clearly up-regulated in the adrenal glands and kidneys of pregnant female rats, whereas the corresponding tissues in nonpregnant rats expressed mainly the immature receptor. Thus, although the gonadal and nongonadal urogenital tissues appear to express the receptor protein constitutively, the functional maturation of the receptors seems to be developmentally and physiologically regulated. It can be hypothesized that the number of fully mature receptors at the cell surface may be up-regulated due to enhanced maturation and cell surface targeting of ER-retained receptors or, alternatively, lengthening the half-life of the mature receptors at the cell surface. The former possibility is more likely based on studies using heterologous expression systems. Namely, in human embryonic kidney 293 cells, newly synthesized LHRs appear to be very prone to premature degradation, and inhibition of degradation was found to lead to enhanced maturation of the ER-retained immature receptors (51). Whether these findings on regulated maturation of LHRs in gonadal and nongonadal tissues can be extended to other mammalian species is not known at present. Interestingly, the human LHR has been reported to be expressed mainly in the mature form in heterologous expression systems (1), in contrast to what has been observed for rat and porcine LHRs (12, 13, 14, 51).
The expression of LHRs in developing rodent urogenital tissues suggests that the receptor might have a functional role in the differentiation of these tissues. Nevertheless, it is unlikely that the immature receptors detected in the developing fetal tissues are fully functional. This idea is suggested by the observation that the immature receptor forms that carry unprocessed N-linked glycans have been found to reside intracellularly in heterologous expression systems (12, 37, 51) and, therefore are unlikely to respond to circulating hormones. It is notable, however, that the immature receptors were capable of hormone binding, because they were purified by ligand affinity chromatography, a finding in line with the results of previous studies (37). This suggests that the hormone-binding sites that have been detected in the developing testes (40) are likely to represent immature nonfunctional receptors. There are some existing in vitro data suggesting that fetal gonads are able to respond to hormonal stimulation (41), but increasing evidence suggests that the LHR might have an insignificant role in intrauterine sex differentiation. For example, recent studies of LHR knockout mice have shown that the newborns are born phenotypically normal, and only postnatal sexual development is impaired (42, 43). In addition, it has been shown that prenatal testosterone levels are normal in hypogonadal (hpg) mice that lack GnRH and, thus, circulating gonadotropins (44). Although one cannot completely rule out the possibility that a few mature receptors are expressed in developing urogenital tissues, our data support the idea that the LHR may have a negligible role in development of the gonadal and nongonadal urogenital tissues. It is also possible that LHRs detected in many other nongonadal tissues represent nonfunctional immature receptors (2, 3, 4, 5). An exception appears to be the nervous tissue that was found to express mature LHR in both adult animals and fetuses (11).
Previous studies have demonstrated LHR expression in rodent and human adrenal glands (7, 8, 9, 45), and the results of the present study support and extend these findings. The transgenic prenatal mice showed ?-Gal activity in developing glands, and receptor expression was verified in fetal and adult rats at the mRNA and protein levels. Receptor expression was low in adults, but was increased substantially during pregnancy. Importantly, pregnant adrenal glands expressed mostly the mature receptor form, suggesting that these receptors are very likely to be fully functional. Both direct and indirect evidence in the literature suggests that this may indeed be the case. For example, human fetal adrenal glands have been shown to respond to hCG administration by increasing dehydroepiandrosterone sulfate secretion (46), and adrenal glands of transgenic female mice expressing bovine LH?-CTP (a chimeric protein derived from the -subunit of bovine LH and a fragment of the ?-subunit of hCG) were found to express LHR and respond to hCG with significantly increased cAMP, progesterone, and corticosterone production (8). In addition, cases of ACTH-independent Cushing’s syndrome have been described, in which adrenocortical hyperfunction was found to be LH/hCG dependent and responsive to treatment with a GnRH analog (26). Whether LH and CG are able to regulate adrenal gland steroid production during pregnancy remains to be tested in the future. In addition, the factor(s) that regulates LHR expression in the adrenal glands and causes up-regulation during pregnancy remains to be identified. A potential candidate, prolactin, is known to up-regulate LHRs in the corpus luteum during pregnancy (47, 48). In rodents, the secretion of pituitary prolactin is known to cease during pregnancy, but there are numerous placentally produced lactogens that are secreted during mid and late pregnancy (49, 50).
In conclusion, our results on LHR expression in rodent gonadal and nongonadal urogenital tissues implicate new and complex mechanisms underlying the regulation of expression of this GPCR. Furthermore, the pregnancy-induced up-regulation of mature LHR expression in female adrenal glands and kidneys predicts novel and previously unanticipated roles for LH and CG in the functional regulation of these tissues. Future studies should explore the mechanisms and define the factors that regulate the expression of mature functional receptors in developing and adult tissues.
Acknowledgments
We are grateful to Dr. Sirpa Kontusaari for generating the transgenic mice, and to Dr. Jussi Tuusa for critical reading of the manuscript. We thank Lissu Hukkanen, Pirkko Peronius, Paula Salmela, and Liisa K?rki for their skillful technical assistance.
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Address all correspondence and requests for reprints to: Dr. Ulla Pet?j?-Repo or Dr. Hannu Rajaniemi, Department of Anatomy and Cell Biology, University of Oulu, P.O. Box 5000, University of Oulu, FI-90014 Oulu, Finland. E-mail: ulla.petaja-repo@oulu.fi or hannu.rajaniemi@oulu.fi.
Abstract
The LH receptor (LHR) is a G protein-coupled receptor involved in the regulation of ovarian and testicular functions. In this study we demonstrate novel and unexpected patterns of receptor expression and regulation in fetal and adult rodent urogenital and adrenal tissues. Two rat LHR promoter fragments (2 and 4 kb) were shown to direct expression of the lacZ reporter in transgenic mice to gonads, adrenal glands, and kidneys, starting at 14.5 d post coitum, and to genital tubercles, starting at 11.5 d post coitum. These tissues were also found to express the full-length LHR mRNA and protein during rat fetal development, but, importantly, only immature receptors carrying unprocessed N-linked glycans were detected. After birth, the receptor gene activity ceased, except in the gonads, which started to express the mature receptor carrying fully processed N-linked glycans. Surprisingly, both LHR mRNA and mature protein levels were up-regulated substantially in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital tissues. Taken together, these results indicate that the LHR protein is expressed constitutively in gonadal and nongonadal urogenital tissues as well in adrenal glands, but its final functional maturation at the posttranslational level appears to be developmentally and physiologically regulated.
Introduction
THE LH RECEPTOR (LHR), like other glycoprotein hormone receptors, the FSH and TSH receptors, belongs to the leucine-rich repeat containing G protein-coupled receptors (GPCRs), with a large extracellular N-terminal domain that is involved in ligand binding (1). This integral membrane protein plays an essential role in the regulation of reproductive functions and is expressed in ovarian granulosa, thecal, interstitial, and luteal cells and in testicular Leydig cells (1). However, it appears to have a wider expression than was previously believed and has also been found to be expressed elsewhere in the reproductive tract (2, 3, 4) and in the placenta (2), umbilical cord (5), prostate (6), adrenal gland (7, 8, 9), and nervous tissue (3, 10, 11). Neither the functional role nor the regulation of nongonadal LHR expression is known at present.
Previous studies have shown that a substantial portion of LHRs in the gonads and nervous tissues as well as in heterologous expression systems exists in an immature form (11, 12, 13, 14), containing high mannose-type N-linked glycans, which are typical for proteins located in the endoplasmic reticulum (ER) (15). This suggests that they might mature inefficiently, a phenomenon that has been found to characterize a few other GPCRs (16, 17, 18, 19, 20). This idea is supported by our recent finding that in stably transfected human embryonic kidney 293 cells, about 80% of newly synthesized rat LHRs is retained in the ER and is targeted for ER-associated degradation (51). Because blockade of the degradation pathway was found to lead to enhanced ER export and maturation of the receptors, LHR expression at the cell surface may be controlled at the ER level. To determine whether maturation of LHRs could be a regulated phenomenon in vivo, we set out to examine the distribution and ontogeny of receptor expression in fetal and adult rodents in more detail, giving special attention to the developing urogenital structures (the term urogenital structures includes gonads, kidneys, genital tubercles, and adrenal glands). To this end, rat LHR promoter-driven transgene activity was assessed in transgenic mice, and receptor expression in fetal and adult rats was determined at the mRNA and protein levels. We demonstrate that during fetal development the receptor is expressed in various urogenital structures: the developing gonads, kidneys, genital tubercles, as well as adrenal glands. However, only immature receptors were detected; mature receptors were found only in adult gonads and, surprisingly, in pregnant female adrenal glands and kidneys. These results thus indicate that the abilities of the different tissues to synthesize and process the LHR protein may be developmentally and physiologically regulated.
Materials and Methods
Promoter constructs and production and analysis of transgenic mice
The construction, production, and analysis of transgenic mice were previously described (11). The rat LHR promoter-driven transgene constructs carried –2060 or –3970 bp from the translation initiation site of the rat LHR gene in front of the reporter gene lacZ. Pregnant females, neonates, and adult mice (60 d) were killed by inhalation of carbon dioxide, and fetuses and tissue samples from adult mice were collected, prefixed, and stained for ?-galactosidase (?-Gal) activity using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (11). After postfixation, the fetuses were observed as whole mounts under a stereomicroscope or, alternatively, were dissected for isolation of urogenital tissues. The paraffin-embedded tissue sections (10 μm) were counterstained with safranin.
Rat tissue preparation
Sprague Dawley rats were used for collecting tissues for RT-PCR and receptor purification. Pregnant females, neonates, and adult rats (90 d old) were killed, and tissue samples were removed, frozen in liquid nitrogen, and stored at –80 C. Dissection of fetal tissues was performed under a stereomicroscope. The use of animals was approved by the University of Oulu committee for the care of experimental animals.
RT-PCR
Four oligonucleotide primers of 29–36 nucleotides were designed based on the rat LHR sequence (GenBank accession no. M26199) (21), as previously described (11). Primers 1 (nucleotides –27 to +9 from translation initiation site) and 2 (nucleotides +2116 to +2084) were used for the RT-PCR, and primers 3 (nucleotides +65 to +97) and 4 (nucleotides +1887 to +1858) were used for the subsequent nested PCR. The expected molecular size of the nested PCR-amplified cDNA product was 1.9 kb, slightly smaller than the original full-length cDNA of 2.1 kb.
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA), according to the manufacturer’s instructions. The RT-PCR was carried out as described previously (11) in a total volume of 50 μl, using 1 μg total RNA as template and 0.2 μM primers 1 and 2. For the second amplification round, 1 μl of the product from the first amplification was used as a template with the slightly intended primers 3 and 4 in the same PCR conditions as in the first round. The PCR products (20 μl) were analyzed on a 1% agarose gel containing ethidium bromide.
Immunoprecipitation, ligand affinity chromatography, and Western blotting
Total cellular membranes were prepared by homogenizing with a Polytron homogenizer (Ultra-Turrax T-25, Janke & Kunkel, Staufen, Germany) in buffer A (PBS, pH 7.5, containing 5 mM EDTA, 5 mM N-ethylmaleimide, and 0.2 mM phenylmethylsulfonylfluoride; 100 mg/ml) and centrifuging at 100 x g for 10 min. The membrane particles were then collected by centrifuging the supernatant for 30 min at 45,000 x g. After washing with buffer A, the protein concentration was determined by Bio-Rad’s DC assay kit, using BSA as the standard. Tissue samples (10–60 mg/sample) from 20–30 fetuses [15.5–18.5 d post coitum (dpc)] and four to eight 1-d-old female or male neonates were pooled for membrane preparation. Membranes from the fetuses, neonates, and adult male testes (8 mg) and 15.5 and 19.5 dpc pregnant (1 mg) and nonpregnant (0.5 mg) female ovaries, adrenal glands (1 mg), and kidneys (6 mg) were then solubilized by stirring on ice for 60 min in 500 μl buffer B [buffer A containing 0.5% (wt/vol) Triton X-100 and 20% (vol/vol) glycerol] and centrifuging at 100,000 x g for 60 min. The soluble fraction was supplemented with 0.1% (wt/vol) BSA and subjected to immunoprecipitation or human chorionic gonadotropin (hCG) affinity chromatography, and the purified samples were analyzed by Western blotting, as described previously (11).
Results
LHR promoter-driven transgene expression in urogenital structures of developing and adult mice
To demonstrate LHR expression in the fetal and adult rodent urogenital structures, we first assessed LHR promoter activity in transgenic mice, which carry the Escherichia coli lacZ gene under control of the rat LHR promoter fragments of 2060 and 3970 bp (11). Both promoter fragments directed expression of the LacZ gene to the urogenital structures in a similar manner, and thus, a representative mouse line expressing the lacZ gene under the 3970-bp LHR promoter was chosen for additional analyses.
Expression of the lacZ gene was examined by whole mount staining of the fetal urogenital blocks or adult tissues with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside. Paraffin-embedded tissue sections (10 μm) were also examined to assess the transgene expression at the cellular level. As shown in Fig. 1A, ?-Gal activity was first detectable in the developing gonads in the 14.5-dpc fetuses, whereas no activity was apparent in the gonads of nontransgenic littermates (Fig. 1B). In the developing ovaries, ?-Gal activity remained at a low level (Figs. 1C and 2, A and B) until in the nearly fertile 30-d-old mice, in which distinct staining in the developing follicles was detected (Fig. 2C). In the adult females, transgene activity was clearly apparent in the maturing ovarian follicles and corpora lutea (Fig. 2D) and was located in granulosa, thecal, and luteal cells and in a varying degree to the ova and interstitial glandular cells (Fig. 2M). The corresponding nontransgenic littermates showed no staining (Figs. 1D and 2, E and F). Some specific transgene activity was also detected in the adult female uterus, but was not studied further. In the developing testes, specific ?-Gal expression was detected in the interstitial tissue, but not in the tubules (Figs. 1, E and F, and 2, G–L), and was clearly restricted to Leydig cells in adult testes (Fig. 2N). ?-Gal activity in the epididymis and ductus deferens was due to endogenous enzyme activity (Fig. 2, compare G to K and J to L), and expression of the transgene in these tissues could therefore not be examined.
FIG. 1. Expression of the LHR promoter-driven transgene in pre- and neonatal mouse urogenital structures. Dissected urogenital blocks of transgenic fetuses at age 14.5 dpc (A) and 16.5 dpc (C and E), and adrenal glands from 1-d-old neonates (G) were analyzed in situ for ?-Gal activity of the rat LHR promoter-LacZ construct, as described in Materials and Methods. The corresponding nontransgenic samples are presented in B, D, F, and H. In the 14.5-dpc mice (A), expression was detected in developing gonads, adrenal glands, and kidneys. The same expression pattern was detected in the corresponding structures of the 16.5-dpc transgenic females (C) and males (E), but in a more dispersed manner. Adrenal glands of 1-d-old neonates (G) showed distinct staining. Ad, Adrenal gland; Go, gonad; Ki, kidney; Ov, ovary; Sg, sensory ganglion; Te, testis; Tg, transgenic; Wt, wild type.
FIG. 2. Expression of the LHR promoter-driven transgene in postnatal mouse gonads. Dissected gonads were stained as described in Fig. 1. The dispersed staining observed in the ovaries of transgenic 1-d-old (A) and 15-d-old (B) females became more intense in those of 30-d-old (C) and adult (D) females, in which ?-Gal activity was clearly apparent in developing follicles and corpora lutea (active and inactive corpora lutea are shown by arrowhead and arrow, respectively). Distinct staining in the testicular interstitial tissue could be seen in transgenic 1-d-old (G), 15-d-old (H), 30-d-old (I), and adult (J) males. Ovaries of nontransgenic 1-d-old (E) and adult female (F) mice showed no expression. The same was true for the testes of nontransgenic males (K and L), but the epididymis displayed endogenous ?-Gal activity. The paraffin-embedded transgenic adult ovaries (M) and testes (N) were sectioned (10 μm) and counterstained with safranin, as described in Materials and Methods. ?-Gal staining could be detected in ovarian interstitial tissue, in thecal and granulosa cells (arrow) of the developing follicles (M), and in testicular Leydig cells (arrow; N). Scale bars, 120 μm (M) and 20 μm (N). Ep, Epididymis; Se, seminiferous tubule; Tg, transgenic; Wt, wild type.
In the 14.5-dpc transgenic fetuses (Fig. 1A), ?-Gal activity could be detected not only in the gonads, but, surprisingly, also in the developing adrenal glands and kidneys. Branched tree-like expression in the kidneys was localized to the tubular epithelium, whereas in the adrenal glands, enzyme activity was detected in mesenchymal cells. Similar staining was detected in 16.5-dpc female (Fig. 1C) and male (Fig. 1E) fetuses. In 1-d-old neonates, distinct staining was found in the medulla and cortex of transgenic (Fig. 1G), but not nontransgenic (Fig. 1H), adrenal glands, after which it became undetectable. The medullary staining is most likely due to transgene expression in the ganglion cells that are of the same origin as those in sympathetic ganglia (11). In kidneys, endogenous ?-Gal activity was apparent in 1-d-old neonates, and the transgene and endogenous expression could therefore not be separated after birth.
Whole mount staining of the transgenic mouse fetuses revealed that the LHR promoter-driven transgene was also expressed in genital tubercles and paired genital swellings, representing the developing external genitalia. The expression was apparent in genital tubercles in the 11.5-dpc fetuses (Fig. 3A) and from 14.5 dpc onward also in the urogenital fold (Fig. 3C, arrow). At the cellular level, expression was localized to the outmost mesenchyme of the genital tubercle (Fig. 3K). In the 1-d-old neonates, ?-Gal activity was clearly apparent in the external genitalia in both females and males (Fig. 3, G and I, respectively), and no distinct sex-dependent differences were observed, although there was a clear difference in the anatomical shape of the corresponding structures. The nontransgenic littermates showed no ?-Gal staining (Fig. 3, D, F, H, J, and L).
FIG. 3. Expression of the LHR promoter-driven transgene in pre- and neonatal mouse genital tubercles. Dissected genital tubercles of transgenic fetuses aged 11.5 dpc (A), 13.5 dpc (B), 14.5 dpc (C), and 16.5 dpc (E) and from a 1-d-old neonatal female (G) and male (I) were stained as described in Fig. 1. The nontransgenic genital tubercles of 14.5- and 16.5-dpc fetuses and 1-d-old neonatal females and males are presented in D, F, H, and J, respectively. The arrows in C and E indicates the urogenital fold, and the arrows in G and H show the paired genital tubercle. The paraffin-embedded 14.5-dpc fetal transgenic (K) and nontransgenic (L) genital tubercles were sectioned (10 μm) and counterstained with safranin. Distinct ?-Gal staining could be detected in the outermost mesenchymal cell layer in the transgenic, but not nontransgenic, genital tubercles. Scale bars, 100 μm (K and L). Tg, Transgenic; Wt, wild type.
Taken together, these results indicate that during organogenesis the LHR promoter is first activated in the developing genital tubercles around 11.5 dpc and in the gonads, adrenal glands, and kidneys at around 14.5 dpc. After birth, activity decreases in adrenal glands and kidneys, but increases substantially in gonads at puberty, residing in specific cell types in mature ovaries and testes.
LHR mRNA expression in urogenital structures of developing and adult rats
To examine LHR expression in the developing urogenital structures, total RNA was isolated from fetal and adult rat tissues and subjected to RT-PCR, followed by nested PCR for amplification of the products. The primers used were selected to encode the entire coding region of the LHR (11). As expected, two distinct amplified products of 2.1 and 1.8 kb were detected after the first amplification round in adult testes and ovaries (Fig. 4A, upper panel, lanes 3 and 6, respectively), corresponding to the full-length LHR and the most common receptor variant, respectively (22, 23, 24). Occasionally, a third transcript of 2.0 kb was detected, most likely representing a hybrid of the 1.8- and 2.1-kb forms (3). In the second amplification round, the final products were slightly smaller than the original cDNA due to the nested primers used (Fig. 4A, lower panel, lanes 3 and 6). As shown in lanes 1 and 4 in Fig. 4A, the full-length transcripts were present in the testes as well as in the ovaries of 17.5-dpc fetuses, the youngest age studied, as well as in 1-d-old neonates (lanes 2 and 5). In developing testes, the transcripts were apparent after the first amplification round, but required additional amplification in the ovaries. The two major mRNA products were also obtained from fetal and neonatal adrenal glands (Fig. 5A, lanes 1–4) and kidneys (Fig. 5A, lanes 7–10) as well as from genital tubercles (data not shown). However, as in fetal and neonatal ovaries, the LHR mRNA transcripts were detected only after the second amplification round. When RNA from adult female adrenal glands was used as a template, the transcripts were detectable after the first amplification round (Fig. 5A, lane 5), whereas the male samples revealed transcripts only after the second round (Fig. 5A, lane 6). The adult kidneys yielded no amplification products (Fig. 5A, lanes 11 and 12). Taken together, these results suggest that the developing urogenital structures start to express full-length LHR mRNA early during differentiation. Although there seem to be no differences in the onset of mRNA expression in males and females, the amount of transcripts appears be higher in males, with the exception of the adrenal glands.
FIG. 4. Expression of LHR mRNA and protein in rat testes and ovaries. A, RT-PCR analyses of the total RNA from testes (lanes 1–3) and ovaries (lanes 4–7). The analyses were performed with primers flanking the entire coding region of the LHR to amplify the full-length cDNAs (upper panel), followed by nested PCR with the intended primers (lower panel), as described in Materials and Methods. Ovarian RNA without RT reaction (lane 7) was used as a negative reaction control. Lanes 1 and 4, 17.5 dpc; lanes 2 and 5, 1-d-old; lanes 3, 6, and 7, adult. The major amplified mRNA products are indicated on the left. B, Western blots of purified receptors from testes (lanes 1–3, 7, and 8) and ovaries (lanes 4–6, 9, and 10). Solubilized cellular membranes from the 15.5- to 18.5-dpc (lanes 1 and 4), 1-d-old (lanes 2, 5, 7, and 9), and adult (lanes 3, 6, 8, and 10) rat gonads were subjected to immunoprecipitation or ligand affinity chromatography and analyzed by Western blotting using a polyclonal antibody directed against the C-terminus of the receptor, as described in Materials and Methods. The mature receptor forms are indicated by arrows, and the immature ones are shown by arrowheads. *, Ig heavy chains. Mr markers are indicated on the right and left.
FIG. 5. Expression of LHR mRNA and protein in rat adrenal glands and kidneys. A, RT-PCR analyses of the total RNA from adrenal glands (lanes 1–6) and kidneys (lanes 7–12). RT-PCR and the subsequent nested PCR were performed as described in Materials and Methods. Lanes 1 and 7, 16.5-dpc female; lanes 2 and 8, 16.5-dpc male; lanes 3 and 9, 1-d-old female; lanes 4 and 10, 1-d-old male; lanes 5 and 11, adult female; lanes 6 and 12, adult male. B, Western blots of purified receptors from adrenal glands. Solubilized cellular membranes from the adrenal glands of 15.5- to 18.5-dpc fetuses (lane 1), 1-d old females (lanes 2 and 6), 1-d-old males (lanes 3 and 7), adult females (lanes 4 and 8), and adult males (lanes 5 and 9) were subjected to immunoprecipitation or ligand affinity chromatography and analyzed by Western blotting, as described in Materials and Methods. *, Ig heavy chains.
LHR protein expression in urogenital structures of developing and adult rats
To determine whether the full-length receptor mRNA detected in the developing urogenital structures is translated to protein, Triton X-100-solubilized membranes were subjected to immunoprecipitation using an LHR-specific polyclonal antibody directed against the C-terminal domain of the protein (25). The immunoprecipitated samples were then analyzed by Western blotting using the same antibody for detection. As we have previously documented (11), two receptor species with apparent molecular masses (Mr) of 90,000 and 73,000 were detected from the adult rat ovary (Fig. 4B, lane 6). These species were shown to carry complex-type and high-mannose-type N-linked oligosaccharides, respectively (11), thus representing mature and immature forms of the receptor. Two receptor species were also immunoprecipitated from the adult testes (Fig. 4B, lane 3), but the larger species migrated more slowly (Mr, 100,000), most likely due to tissue-specific differences in the glycan moieties. Importantly, both the mature and immature receptor species were purified by ligand (hCG) affinity chromatography (Fig. 4B, lanes 8 and 10), showing that they were able to bind hormone.
Surprisingly, none of the developing or adult nongonadal urogenital tissues were found to express the mature LHR, except for adult female adrenal glands, in which the mature receptor was just barely detectable (Fig. 5B, lanes 4 and 8). Only the 73,000 Mr receptor species was immunoprecipitated from fetal gonads (Fig. 4B, lanes 1 and 4), adrenal glands (Fig. 5B, lane 1), and kidneys (data not shown) as well as from 1-d-old female ovaries (Fig. 4B, lane 5), adrenal glands (Fig. 5B, lane 2), and kidneys (data not shown), whereas two receptor species of 73,000 and 70,000 Mr were purified from the corresponding 1-d-old male tissues (testes: Fig. 4B, lanes 2; adrenal gland: Fig. 5B, lane 3). These two receptor species were also purified from female and male fetal and neonatal genital tubercles (data not shown). Both the 73,000 and 70,000 Mr receptor forms were also detected by a polyclonal antibody that was raised against the N-terminal domain of the rat LHR (25), ruling out the possibility that the 70,000 Mr species might represent a degradation product of the larger one.
In summary, these results demonstrate that a mature LHR species carrying fully processed N-linked oligosaccharides appears to be expressed only in the gonads of adult rats and to some extent in the adult female adrenal glands, whereas only immature receptor forms are expressed in fetal and neonatal rat urogenital structures. This apparent tissue- and age-dependent difference in the ability of cells to process the LHR protein to the mature form may thus be developmentally and physiologically regulated.
LHR expression in adrenal glands and kidneys of pregnant rats
The results presented above suggest that the LHR is expressed in fetal adrenal glands and kidneys and to some extent also in adult female adrenal glands. This finding together with the previous observation that receptor expression in adrenal glands may be hormonally regulated (8, 26) prompted us to determine whether LHR expression might be up-regulated during pregnancy. To this end, tissue samples from pregnant female rats of 15.5 and 19.5 dpc were isolated and analyzed. As expected, the mRNA expression level in the ovaries was higher in pregnant females compared with nonpregnant animals (compare Fig. 6A, lanes 1 and 2, to Fig. 4A, lane 6). Importantly, mRNA expression was also substantially higher in the adrenal glands and kidneys. The transcripts were clearly detectable at 15.5 dpc after the first amplification round (Fig. 6A, lanes 3 and 5, respectively), which was in clear contrast with the findings obtained for nonpregnant females (see Fig. 5A, lanes 5 and 11). However, unlike in the ovaries, expression decreased at 19.5 dpc (Fig. 6A, lanes 4 and 6) and was detected in the kidneys only after the second amplification round (data not shown).
FIG. 6. Expression of LHR mRNA and protein in ovaries, kidneys, and adrenal glands of pregnant female rats. A, RT-PCR analyses of the total RNA from ovaries (lanes 1 and 2), adrenal glands (lanes 3 and 4), and kidneys (lanes 5 and 6). The analyses were performed as described in Materials and Methods. Lanes 1, 3, and 5, 15.5-dpc pregnant females; lanes 2, 4, and 6, 19.5-dpc pregnant females. B, Western blots of immunoprecipitated (lanes 1–3) or ligand affinity purified (lanes 4–6) receptors from pregnant (15.5 dpc) female ovaries (lanes 1 and 4), adrenal glands (lanes 2 and 5), and kidneys (lanes 3 and 6). The samples were analyzed as described in Materials and Methods. *, Ig heavy chains.
To determine whether the high amount of mRNA transcripts detected in the adrenal glands and kidneys during pregnancy might correlate to the expression of mature receptors in these tissues, samples were prepared for immunoprecipitation. As shown in Fig. 6B, the LHR-specific antibody recognized two receptor species, of 90,000 and 73,000 Mr, from the ovaries (lane 1) as well as the adrenal glands (lane 2) and kidneys (lane 3) of 15.5-dpc pregnant females. Both of these receptor species were purified by ligand affinity chromatography (Fig. 6B, lanes 4–6), indicating that they are capable of hormone binding. Near the time of delivery, at 19.5 dpc, the expression decreased to approximately the same level as in nonpregnant females, and the adrenal glands and kidneys again appeared to contain mostly the immature LHR form (data not shown). Only the adrenal gland displayed a small amount of receptor corresponding to the mature form.
Thus, these results demonstrate that although the immature LHRs are the predominant receptor species detected in adult female rat adrenal glands, there is a clear shift to an increased level of mature receptors during pregnancy in both adrenal glands and kidneys. This finding suggests that LHR expression may be hormonally regulated in these nongonadal tissues.
Discussion
In the present study we demonstrate that the LHR is expressed not solely in the gonads, but is also present in other urogenital structures, such as kidneys and genital tubercles as well as adrenal glands. The receptor gene activity appears to start early during fetal development, but importantly, the receptor protein is expressed only in an immature form. Mature receptors were detected only in adult gonads and, surprisingly, in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital structures. These results implicate new and complex roles for LH and CG and indicate that the expression of mature and functional LHRs is developmentally and physiologically regulated in both gonadal and nongonadal urogenital tissues.
The tissue- and age-dependent expression of the LHR in urogenital structures was first examined using transgenic mice carrying the lacZ reporter under the rat LHR promoter. The two promoter fragments of 2060 and 3970 bp used directed ?-Gal expression in a similar manner to developing and adult tissues, and as expected, the reporter activity was detected in specific gonadal cells in adult mice, testicular Leydig cells, and ovarian thecal, granulosa, and luteal cells. In the developing fetuses, reporter gene activity was first detected in the gonads at 14.5 dpc, concomitantly with the adrenal glands and kidneys, whereas the genital tubercles showed activity even earlier at 11.5 dpc. In contrast to these findings, H?m?l?inen and co-workers (9, 27, 28) were unable to detect transgene expression in prenatal transgenic mice when mouse LHR promoter fragments of 7.4, 2.1, or 173 bp were used. Furthermore, the mouse promoter fragments were found to show clear sex- and tissue-dependent differences in activity in adult tissues. The reasons for these differences are not known, but they may be due to species differences or differences in the length and usage of the 5'-flanking sequences of the two genes.
The findings obtained with transgenic mice were substantiated by demonstrating LHR expression in various rat urogenital structures at the mRNA level. Two distinct mRNA species of 2.1 and 1.8 kb, corresponding to the full-length LHR and the most common variant, respectively (22, 23, 24), were amplified from fetal and adult rat tissues; importantly, no apparent sex differences in the onset of mRNA expression were observed. For example, the full-length mRNA transcripts were detected in the gonads at 17.5 dpc in both female and male fetuses. This is in contrast with previous findings that rat ovaries were found to express only truncated mRNA species before birth (29). These apparent differences are probably due to the low amount of full-length receptor transcripts in the developing ovaries that may have hindered their detection previously, especially because different techniques to detect mRNA expression were used. In the present study the transcripts were detectable in fetal ovaries by RT-PCR only after the second amplification round, whereas those in the testes were easily detectable after the first round. This sex-dependent difference in the amount of receptor transcripts in the fetal gonads was not unexpected, however, because the same difference was clearly apparent in adult gonads as well.
The LHR expression in developing and adult urogenital tissues was also demonstrated at the protein level. Immunoprecipitation using a receptor-specific antibody revealed two receptor species of 90,000 and 73,000 Mr from adult rat ovaries and pregnant rat adrenal glands and kidneys, whereas 100,000 and 73,000 Mr species were obtained from adult testes. Similar receptor species have been found previously in adult rat and porcine gonads (12, 13, 30, 31, 32, 33, 34, 35) and in fetal and adult rat nervous tissue (11) as well as in various heterologous expression systems (12, 14, 51). The 90,000 and 100,000 Mr receptor forms correspond to fully mature receptors, because they have been shown to contain complex type N-linked oligosaccharides, and their size difference is due to tissue-specific glycosylation (11, 12, 13, 14, 33, 36). The 73,000 Mr species, in contrast, is likely to represent immature receptor precursors, because it carries high-mannose-type N-linked glycans (11, 12, 13, 14, 37), which are typical for glycoproteins residing in the ER. Unlike in the adults, fetal urogenital tissues were found to express not mature receptors, but only two smaller species of 73,000 and 70,000 Mr. Because both of these forms were recognized by antibodies directed against the N- and C-terminal domains of the receptor, the smaller species does not represent a degradation product of the larger one, but, instead, may be an intact receptor precursor with a fewer number of N-linked glycans than the 73,000 Mr form. The scarcity of the tissue samples prevented us from testing this possibility in more detail. Alternatively, the 70,000 Mr receptor form might result from alternative splicing of the primary transcript and represent a receptor isoform lacking some of the exons coding for the extracellular domain of the receptor (22, 23, 24). This is an interesting possibility, because alternatively spliced transcripts are especially abundant in male gonads (38, 39), in which the 70,000 Mr receptor species was the most abundant one.
It has been suggested that LHR expression in the developing gonads is regulated at a posttranscriptional level, because the onset of expression of receptors capable of hormone binding in the developing testes and ovaries has been reported to coincide with the change in the alternative splicing pattern of the LHR mRNA (29, 39). Our results extend these findings and argue that the expression of fully functional mature receptors is also regulated at a posttranslational level in both gonads and nongonadal urogenital tissues. This idea was supported by the fact that there was a clear age-, tissue-, and sex-dependent variation in the relative amount of mature receptors to immature ones. Furthermore, the expression of mature receptors was clearly up-regulated in the adrenal glands and kidneys of pregnant female rats, whereas the corresponding tissues in nonpregnant rats expressed mainly the immature receptor. Thus, although the gonadal and nongonadal urogenital tissues appear to express the receptor protein constitutively, the functional maturation of the receptors seems to be developmentally and physiologically regulated. It can be hypothesized that the number of fully mature receptors at the cell surface may be up-regulated due to enhanced maturation and cell surface targeting of ER-retained receptors or, alternatively, lengthening the half-life of the mature receptors at the cell surface. The former possibility is more likely based on studies using heterologous expression systems. Namely, in human embryonic kidney 293 cells, newly synthesized LHRs appear to be very prone to premature degradation, and inhibition of degradation was found to lead to enhanced maturation of the ER-retained immature receptors (51). Whether these findings on regulated maturation of LHRs in gonadal and nongonadal tissues can be extended to other mammalian species is not known at present. Interestingly, the human LHR has been reported to be expressed mainly in the mature form in heterologous expression systems (1), in contrast to what has been observed for rat and porcine LHRs (12, 13, 14, 51).
The expression of LHRs in developing rodent urogenital tissues suggests that the receptor might have a functional role in the differentiation of these tissues. Nevertheless, it is unlikely that the immature receptors detected in the developing fetal tissues are fully functional. This idea is suggested by the observation that the immature receptor forms that carry unprocessed N-linked glycans have been found to reside intracellularly in heterologous expression systems (12, 37, 51) and, therefore are unlikely to respond to circulating hormones. It is notable, however, that the immature receptors were capable of hormone binding, because they were purified by ligand affinity chromatography, a finding in line with the results of previous studies (37). This suggests that the hormone-binding sites that have been detected in the developing testes (40) are likely to represent immature nonfunctional receptors. There are some existing in vitro data suggesting that fetal gonads are able to respond to hormonal stimulation (41), but increasing evidence suggests that the LHR might have an insignificant role in intrauterine sex differentiation. For example, recent studies of LHR knockout mice have shown that the newborns are born phenotypically normal, and only postnatal sexual development is impaired (42, 43). In addition, it has been shown that prenatal testosterone levels are normal in hypogonadal (hpg) mice that lack GnRH and, thus, circulating gonadotropins (44). Although one cannot completely rule out the possibility that a few mature receptors are expressed in developing urogenital tissues, our data support the idea that the LHR may have a negligible role in development of the gonadal and nongonadal urogenital tissues. It is also possible that LHRs detected in many other nongonadal tissues represent nonfunctional immature receptors (2, 3, 4, 5). An exception appears to be the nervous tissue that was found to express mature LHR in both adult animals and fetuses (11).
Previous studies have demonstrated LHR expression in rodent and human adrenal glands (7, 8, 9, 45), and the results of the present study support and extend these findings. The transgenic prenatal mice showed ?-Gal activity in developing glands, and receptor expression was verified in fetal and adult rats at the mRNA and protein levels. Receptor expression was low in adults, but was increased substantially during pregnancy. Importantly, pregnant adrenal glands expressed mostly the mature receptor form, suggesting that these receptors are very likely to be fully functional. Both direct and indirect evidence in the literature suggests that this may indeed be the case. For example, human fetal adrenal glands have been shown to respond to hCG administration by increasing dehydroepiandrosterone sulfate secretion (46), and adrenal glands of transgenic female mice expressing bovine LH?-CTP (a chimeric protein derived from the -subunit of bovine LH and a fragment of the ?-subunit of hCG) were found to express LHR and respond to hCG with significantly increased cAMP, progesterone, and corticosterone production (8). In addition, cases of ACTH-independent Cushing’s syndrome have been described, in which adrenocortical hyperfunction was found to be LH/hCG dependent and responsive to treatment with a GnRH analog (26). Whether LH and CG are able to regulate adrenal gland steroid production during pregnancy remains to be tested in the future. In addition, the factor(s) that regulates LHR expression in the adrenal glands and causes up-regulation during pregnancy remains to be identified. A potential candidate, prolactin, is known to up-regulate LHRs in the corpus luteum during pregnancy (47, 48). In rodents, the secretion of pituitary prolactin is known to cease during pregnancy, but there are numerous placentally produced lactogens that are secreted during mid and late pregnancy (49, 50).
In conclusion, our results on LHR expression in rodent gonadal and nongonadal urogenital tissues implicate new and complex mechanisms underlying the regulation of expression of this GPCR. Furthermore, the pregnancy-induced up-regulation of mature LHR expression in female adrenal glands and kidneys predicts novel and previously unanticipated roles for LH and CG in the functional regulation of these tissues. Future studies should explore the mechanisms and define the factors that regulate the expression of mature functional receptors in developing and adult tissues.
Acknowledgments
We are grateful to Dr. Sirpa Kontusaari for generating the transgenic mice, and to Dr. Jussi Tuusa for critical reading of the manuscript. We thank Lissu Hukkanen, Pirkko Peronius, Paula Salmela, and Liisa K?rki for their skillful technical assistance.
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