Direct Luteinizing Hormone Action Triggers Adrenocortical Tumorigenesis in Castrated Mice Transgenic for The Murine Inhibin {alpha}-Subunit Promoter/Simian Virus 40 T-Antigen Fusion Gene

Rilianawati, Tommi Paukku, Jukka Kero, Fu-Ping Zhang, Nafis Rahman, Kirsi Kananen and Ilpo Huhtaniemi

Department of Physiology University of Turku 20520 Turku, Finland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transgenic (TG) mice, expressing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin {alpha}-subunit promoter (inh{alpha}p), develop gonadal tumors of granulosa/theca or Leydig cell origin. We showed previously that adrenocortical tumors develop if the TG mice are gonadectomized but never develop in intact animals. However, if functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding the TG mice into the hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumors appeared. Since the most obvious difference between the gonadectomized and GnRH-antagonist-treated or Tag/hpg double mutant mice is the elevated gonadotropin secretion in the first group, we examined whether the adrenal tumorigenesis would be gonadotropin-dependent. Surprisingly, both the adrenal tumors and a cell line (C{alpha}1) derived from one of them expressed highly functional LH receptors (LHR), as assessed by Northern hybridization, immunocytochemistry, ligand binding, and human CG (hCG)-stimulated cAMP and steroid production. No FSH receptor expression was found in the adrenal tumors by RT-PCR. hCG treatment of the C{alpha}1 cells stimulated their proliferation, as measured by [3H]thymidine incorporation. This effect was related to hCG-stimulated steroidogenesis since progesterone, testosterone, and estradiol, at physiological concentrations, also stimulated the C{alpha}1 cell proliferation. Different adrenocortical cells expressed initially LHR and Tag, whereas both were highly expressed in the tumor cells. In conclusion, the high level of functional LHR in the adrenal tumors indicates that this receptor can function as tumor promoter when ectopically expressed and stimulated by the ligand hormone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have produced a transgenic (TG) mouse model for gonadal tumorigenesis using a 6-kb fragment of the mouse inh{alpha}p fused with the Tag coding sequences (1, 2, 3). The gonadal tumors, originating from granulosa/theca or Leydig cells, appear in two established TG mouse lines (IT6-M and IT6-F) with 100% penetrance by the age of 5–8 months. The tumor growth was clearly gonadotropin-dependent (4), in which sense our model resembles the inhibin-{alpha} knock-out mice, which also develop gonadotropin-dependent gonadal tumors (5, 6, 7). TG mice gonadectomized before puberty developed adrenal gland tumors, which was never detected in intact TG mice, suggesting that some gonadal or gonad-dependent factors inhibit the adrenal tumorigenesis in the intact TG mice (3).

The adrenal expression of the endogenous inhibin-{alpha} and transgenic Tag genes were found to be suppressed by inhibin, when tested in cell lines derived from the TG tumors (3). This finding indicated a novel autoregulatory mechanism of inhibin gene expression in the mouse adrenal gland and is in line with its tumor suppressor role as established in inhibin-deficient knock-out mice (5). Since the gonadal tumors produced high concentrations of inhibin, we considered this an important reason for the absence of adrenal tumors in intact mice (3). We therefore assumed that adrenal tumorigenesis could also be induced in TG mice if they are functionally gonadectomized by treatment with a GnRH antagonist or by cross-breeding them into the genetic background of the gonadotropin-deficient hypogonadal hpg mutant mouse (8). However, no adrenal tumors were found in either of these models despite their dramatically suppressed inhibin secretion (4). This prompted us to rule out the role of gonadal inhibin as the suppressor of adrenal tumorigenesis in intact TG mouse. We therefore hypothesized that it could be related to the elevated gonadotropin secretion, which is the most obvious difference between the surgical and functional gonadectomy models. The present findings demonstrate that the adrenal cortex of the TG mice contains LHR which, when coexpressed with Tag, triggers tumorigenesis in the presence of high LH stimulation, as occurs after surgical gonadectomy.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of SB-75 Treatment and the Genetic hpg Background on Adrenal Tumorigenesis of TG Mice
We reported recently the suppression or total inhibition of gonadal tumorigenesis in TG mice treated with SB-75 or those with the genetic hpg background (4). The gonadotropin, testosterone, and inhibin levels were profoundly suppressed by both treatments. Macroscopically visible adrenal gland tumors were not detected in any of the intact control or SB-75-treated female and male TG mice, or in the Tag/hpg double-mutant mice (for n values, see Materials and Methods). In accordance, only marginal differences were observed between the adrenal gland weights of the different treatment groups (results not shown). In contrast, all surgically gonadectomized male and female TG mice, so far studied in our laboratory (>50), have developed adrenal tumors at the age of 6–8 months.

LH Receptor (LHR) Expression in Adrenal Tumor Cells and Tissue
The first piece of evidence for LHR expression in the adrenal tumors is provided by Northern hybridization. Clear LHR message with similar proportions of the different splice variants was detected in the adrenal tumors and C{alpha}1 cells, which was very similar to that of the mouse testis, used as positive control (Fig. 1Go). No specific hybridization was observed in the non-TG control adrenals. Even a sensitive RT-PCR method was unable to demonstrate LHR mRNA in adrenal glands of intact and gonadectomized wild-type mice, of both sexes, when studied at the age of 19 days, 2 months, and 5 months (result not shown). RT-PCR of intact TG mice detected, in agreement with the receptor- binding measurements (see below), LHR mRNA in adrenal glands at the age of 6 months, but not in younger animals (results not shown).



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Figure 1. Northern Hybridization Analysis of LHR mRNA in Control Mouse Adrenal Tissue (lane A), Control Testis Tissue (lane B), Male Mouse Adrenal Tumor (lane C), Female Mouse Adrenal Tumor (lane D), and C{alpha}1 Cells (lane E)

Each lane contains 20 µg of total RNA. The migration of the 18S and 28S rRNAs are depicted on the left, and the sizes of the different LHR mRNA splice variants (in kilobases) on the right.

 
Scatchard analysis with [125I]iodo-hCG label demonstrated a high number of LHR in the C{alpha}1 cells (8,000 receptors per cell). However, it is lower than that measured in the BLT-1 murine Leydig tumor cells (47,000 receptors per cell) used as positive control. The equilibrium dissociation constant (Kd) of hCG binding to the adrenal cell receptors was similar to that detected in the Leydig tumor cells (6.8 pmol/liter vs. 22 pmol/liter) (Fig. 2Go).



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Figure 2. Scatchard Analysis of [125I]iodo-hCG Binding to the Adrenal Tumor Cells (C{alpha}1, panel A) and Control Leydig Tumor Cells (BLT-1, panel B)

Both cell types possessed high-affinity LHR with Kd values of 6.8 pmol/liter for C{alpha}1 cells and 22 pmol/liter for BLT-1 cells. The number of binding sites was 8,000/cell for C{alpha}1 cells and 47,000/cell for BLT-1 cells.

 
The adrenal homogenates of the TG and non-TG mice were then studied for specific [125I]iodo-hCG binding (Fig. 3Go). It was clearly detectable in the adrenal tumors of the gonadectomized TG mice, but also, albeit at lower levels, in the adrenal glands of the gonad-intact TG mice. The IT6-F line showed higher binding than the IT6-M TG line, but no sex differences were observed between the LHR levels. Low but significant hCG binding was also observed in the non-TG hpg mice, but not in any of the non-TG mice (Fig. 3Go). In both TG lines, the adrenal LHR persisted after the GnRH-antagonist treatment, albeit at somewhat lower levels (result not shown).



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Figure 3. Single-Point Measurements of [125I]iodo-hCG Binding (fmol/mg protein) to Adrenal Gland Homogenates of Control Female (F) and Male (M) Mice, hpg Mice (HPG), Intact TG Mice (6 month-old) of the IT6-F and IT6-M Lines, Tumorous Adrenals of Gonadectomized Female (T-F) and Male (T-M) TG Mice, and Normal Mouse Testis (Te)

Each bar is the mean + SEM of measurements from three to six tissue samples. The asterisks indicate significant differences from binding measured in the control adrenals, i.e. nonspecific binding (*, P < 0.05; ***, P < 0.001).

 
No FSH receptor expression could be detected in the adrenal tumors or C{alpha}1 cells when examined by RT-PCR (result not shown).

hCG-Stimulated cAMP and Steroid Production of the C{alpha}1 Adrenal Tumor Cells
cAMP production of the C{alpha}1 adrenal tumor cells was stimulated in dose-dependent fashion by hCG, the highest increase occurring between doses 1–10 µg/liter. The maximum stimulation was about 15-fold over the nonstimulated controls (Fig. 4Go). This finding provides clear evidence that the LHR in the adrenal tumor cells are functional. The steroidogenesis of the cells was monitored by production of progesterone, which, rather than corticosterone, was previously found to be the main steroid produced by the C{alpha}1 cells (3). The basal level of progesterone production was high, and a dose-dependent moderate increase, up to 40% (P < 0.01), was observed in response to hCG (Fig. 4Go). As expected, the ED50 of the progesterone response was about 10-fold lower than that of cAMP (1 vs. 8 µg/liter).



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Figure 4. Response of C{alpha}1 Cell cAMP (left panel) and Progesterone (right panel) Production to Stimulation with hCG

Each bar is the mean ± SEM of three parallel incubations. One of three similar experiments is presented. The asterisks indicate significant differences from the control (=0) level (significant differences as in Fig. 3Go).

 
Effect of hCG and Steroid Hormones on DNA Synthesis of the C{alpha}1 Adrenal Tumor Cells
The DNA synthesis of the C{alpha}1 cells was significantly stimulated by treatment with intermediate doses of hCG, as monitored by [3H]thymidine incorporation (Fig. 5Go). Likewise, treatment with progesterone caused a dose-dependent significant increase in [3H]-thymidine incorporation of the same cells (up to 80%, Fig. 5Go). The minimum effective dose of progesterone was 30 nmol/liter. Testosterone had a stimulating effect on C{alpha}1 cell proliferation, up to 2-fold from control, and the lowest effective dose tested was 10 nmol/liter (Fig. 5Go). A stimulating effect was also found with estradiol treatment, and maximum stimulation was found at a dose of 2 nmol/liter (Fig. 5Go).



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Figure 5. The Effect of hCG (A), Progesterone (B), Testosterone (C), and Estradiol (D) on [3H]Thymidine Incorporation into the C{alpha}1 Cells in a 24-h Culture

The incorporation into control (0) cells ranged from 2000–3000 cpm/105 cells, and because of this interassay variation, the effects of the hormonal treatments in the individual experiments were calculated as percents of control (100%). Each panel shows data (mean ± SEM) of three to five independent experiments. The asterisks indicate significant differences from control (significances as in Fig. 3Go).

 
Immunocytochemistry
Before occurrence of clear-cut tumors, the immunoreactivities of Tag and LHR appeared to be almost exclusively localized in different cells of the adrenal cortex of the gonadectomized TG mice (Fig. 6Go). Both types of cells were organized in columnar arrays. The Tag immunoreactivity was clearly localized in the nuclei, whereas that of the LHR was strongest in the periphery of the cells. Although the majority of the two immunoreativities was localized in different cells, the possibility of some of the immunopositive cells expressing both antigens could not be excluded in the serial sections analyzed. In fact, this is evident since the same adrenocortical tumor cell line (C{alpha}1), derived from one of these tumors, was positive both for Tag, as shown before (3), and for the LHR, as show in this study. The tumor growth originated from the juxtamedullary part of the adrenal cortex (3), which indicates the X-zone as the origin of the malignant growth.



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Figure 6. Immunostaining of Two Adjacent Sections of the Adrenal Gland of a Gonadectomized TG Mouse before Apparent Adrenal Tumor Formation

Panel A shows staining using an anti-LHR antiserum, and panel B shows staining using an anti-Tag antiserum. For orientation, the arrow shows the same arteriole in both sections. Bar, 20 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we examined further the unexpected finding that the inh{alpha}p/Tag mice develop adrenal tumors only if they are neonatally gonadectomized (3, 4). The gonadal tumors produced high levels of inhibin, which also suppressed proliferation of the C{alpha}1 cells, derived from one of the adrenal tumors (3). We therefore hypothesized, based on the tumor suppressor function of inhibin (5), that the elimination of this peptide by gonadectomy triggers the adrenal tumorigenesis. To obtain direct proof for this, we performed functional gonadectomy of the TG mice by treating them with a GnRH antagonist or by cross-breeding them into the hypogonadotropic hpg genetic background (4). No gonadal tumors were formed in these mice, demonstrating the gonadotropin dependence of these malignancies. Neither were adrenal tumors found despite the suppressed inhibin levels, which demonstrated that gonadectomy, rather than eliminating a gonadal tumor suppressor, brought about a factor stimulating adrenal tumorigenesis. Since the high gonadotropin levels after removal of gonads are the most obvious difference between the two experimental models, this hormonal change was considered somehow responsible for the appearance of the adrenal tumors.

Quite surprisingly, we found a high level of LHR gene expression in the tumorous adrenal glands. A low level of LHR was also present in the adrenal glands of the intact TG mice, but never in those of the wild-type mice. Although there is circumstantial evidence for LH responsiveness of the mouse adrenal gland (see below), our finding is to our knowledge the first piece of direct evidence for this. That the expression was so high after gonadectomy is surprising, since high LH levels usually down-regulate the cognate receptor expression (9). This suggests that the regulation of the adrenal tumor LHR gene may differ from that in gonads. LH has been shown to stimulate inhibin {alpha} expression (10, 11), which explains why the high postgonadectomy LH levels apparently increased the Tag transgene expression, thereby triggering the adrenal tumorigenesis. The absence of FSH receptor expression in the adrenal tumors rules out a role of FSH in the tumorigenesis, although the concentration of this hormone is also dramatically increased after gonadectomy.

The present findings shed some more light on the existing indirect data on LH responsiveness of the immature mouse adrenal gland. The mouse adrenal cortex consists of three structurally and functionally distinct layers, i.e. zona glomerulosa, fasciculata, and, unlike other mammalian species, the X-zone (12). The existing findings on function of the X-zone are rather confusing. Although never directly demonstrated, several pieces of data suggest that the X-zone may be responsive to LH. It normally disappears during postnatal development, at puberty in males and after the first pregnancy in females, but it survives after neonatal gonadectomy (12, 13, 14). It also disappears after hypophysectomy but can be preserved by LH treatment (13). A theory has been postulated that LH maintains the X-zone whereas testicular androgens, as well as androgens of unknown origin during pregnancy, cause its involution (12). Absence of this effect may explain why we found a low level of LHR in the adult hpg mouse adrenal glands. The role of LH in the ontogeny of the mouse adrenal cortex clearly needs to be revisited.

With regard to the LH responsiveness of the human adrenal cortex, the findings are also scanty and controversial (15, 16). Very recently, Pabon et al. (17) demonstrated LHR mRNA and immunoreactivity in the human adrenal cortex, but the physiological significance of this finding remains open in the absence of functional data. In contrast, several reports exist on LH responsiveness of human adrenocortical tumors (see, e.g. Refs. 18–20). The relevance of these findings with the present TG model is unclear, since human adrenal tumors with LH responsiveness have been speculated to originate from metaplasia of ovarian theca cells or embryologically competent mesenchymal cell to the adrenal gland (21, 22). In contrast, the presence of LHR in the TG mouse adrenals seems to be strictly related to the Tag expression, occurring only in intact and gonadectomized TG mice at the age of about 6 months.

The adrenal LHR and Tag expression of the gonadectomized TG mice did not follow the anatomical zonation before apparent tumor formation. Both immunoreactivities appeared in cord-like radial structures spanning the adrenal cortex. Interestingly, they appeared to be localized mainly in different cells at this stage. The adrenal tumors always originate from the juxtamedullary X-zone (3), and since they express both LHR and Tag in large amounts, both of them are apparently needed for tumorigenesis. The ligand-stimulated LHR functions as a tumor promoter in this case.

The induction mechanism of LHR expression in gonadal steroidogenic cells is still unknown. It is curious that similar transcription factors, e.g. SF-1, WT-1, and DAX-1, participate in induction of the embryonic differentiation of gonadal and adrenal somatic cells (23, 24, 25), but finally different tropic hormones regulate the two cell types in later life, i.e. ACTH and angiotensin II in the adrenal gland, and gonadotropins in gonads. What brings about this difference in cells that were originally under very similar induction mechanisms, remains unknown. The difference may, in fact, be quantitative rather than strictly qualitative, since low-level LHR expression in the adrenal gland, in particular the mouse adrenal X-zone, may be a normal phenomenon (17). The current TG model will be a good tool for further exploration of this matter.

As expected on the basis of the in vivo findings, hCG increased the thymidine incorporation of the C{alpha}1 cell line. Since progesterone, testosterone, and estradiol also displayed similar effects on the tumor cells, the LH-stimulated steroidogenesis may be the crucial growth signal. Of these steroids, progesterone and estradiol were found to be actively produced by the adrenal tumors (3). A role for LH in adrenal tumorigenesis is also possible in normal mice, since certain mouse strains appear to develop adrenal tumors after gonadectomy (26). Since gonadal tumors also appear in TG mice overexpressing LH (27), this hormone seems to have tumor promoter activity if a first hit (in the present model Tag) has sensitized a cell lineage to malignant growth. Hence, the present TG mice provide a good model for genesis of hormone-dependent cancer. Upon expression of a potent oncogen (Tag), a tropic hormone functions as a tumor promoter triggering the malignant growth.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Animals and Treatments
The mice used were TG for a 6-kb inh{alpha}p/Tag as described previously (1, 2). Males and females of lines IT6-M and IT6-F were used. Genotypic hpg mice (8) were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our own vivarium. Genotyping of the mice was carried out from tail DNA by PCR (1, 8, 28). The mice were specific pathogen-free and housed four to six per cage in controlled conditions of light (12 h light, 12 h darkness) and temperature (21 ± 1 C). They were fed with commercial mouse chow SDS RM-3 (Special Diet Service; E, soy-free; Whitham, Essex, UK) and tap water ad libitum. All the procedures using mice were approved by the University of Turku Ethical Committee on Use and Care of Animals.

Gonadectomy
TG mice (12 females, 11 males) of the IT6-M line and non-TG mice (9 females, 9 males) were castrated at the age of 4 weeks. The heterozygous TG animals were derived from matings of TG males of the IT6-M line (1, 2, 3) with DBA/2J or C57Bl/6 females. Avertin anesthesia (29) during surgical procedures and postoperative analgesia (buprenorphine, 3 mg/mouse, ip) were used. The mice were weighed every other week and inspected daily for potential signs of tumorigenesis. The mice were killed at 6–8 months of age (i.e. 5–7 months after gonadectomy).

GnRH Antagonist Treatment
Six males and seven females of the IT6-M TG mouse line and five males and six females of the IT6-F TG mouse line (all TG heterozygous, derived as above) were injected subcutaneously every 84 h with the GnRH antagonist Cetrorelix acetate (SB-75; Asta Medica AG, Frankfurt am Main, Germany) in 5% mannitol. The dosage of SB-75 was 10 mg/kg body weight per injection. The control group receiving 5% mannitol injections consisted of age-matched TG mice (8 males and 12 females of both lines) and SB-75-treated non-TG mice (1 female, 2 males). The treatment was started at the age of 3 months and lasted for 12 wk, and the mice were killed 3 days after the last injection.

hpg Experiment
At the first mating, TG males of both IT6 lines were cross-bred with females heterozygous (HT) for the hpg mutation (HT females) (4). TG males heterozygous for hpg (Tag/HT males), derived from the first mating, were further cross-bred with HT females to create the hypogonadal transgenic (Tag/hpg) mice used in this study (6 males, 4 females). Tag/HT mice (24 of each sex) served as controls. The mice were killed at the age of 6 months. The findings on gonadal tumorigenesis of the GnRH antagonist-treated and hpg mice have been reported previously (4).

At the end of the treatment/follow-up periods, the mice were anesthetized with Avertin (29) and laparotomy was performed. The adrenal glands were collected, weighed, and snap-frozen in liquid nitrogen or fixed in Bouin’s solution for histological analysis.

Cell Line Stimulations
C{alpha}1 cells, derived previously from one of the TG adrenal tumors (3), were used. One day before stimulation, the C{alpha}1 cells were plated on 24-well plates (Greiner, Labortechnik, Frickenhausen, Germany) at a density of 105 cells per well in 0.5 ml of the culture medium, i.e. DMEM/F12 (1:1, with 0.365 g/liter L-glutamine; Life Technologies, GIBCO BRL, Glasgow, Scotland), 10% heat-inactivated FCS (Bioclear, Berks, UK), 4.5 g/liter glucose, 20 mmol/liter HEPES, 0.1 g/liter gentamicin (Biological Industries, Bet-HaEmek, Israel), and 1.25 mg/liter fungizone (GIBCO), and incubated for 24 h. Each experiment was performed with quadruplicate samples and repeated three to five times. The cells were washed with 1 x PBS (PBS, pH 7.4) and incubated for 24 h in 1 ml of DMEM/F12 + 0.1% BSA (Sigma Chemical Co., St.Louis, MO) and 0.2 mmol/liter of 3-isobutyl-1-isometylxanthine (Aldrich-Chemie, Steinheim, Germany) in the presence and absence of different concentrations of hCG (NIH CR-121, 11500 IU/mg, NIH, Bethesda, MD), progesterone, testosterone, or estradiol (all from Sigma). After incubation for 1 h, aliquots of the media were collected for measurement of cAMP by RIA (30, 31). After incubation for 8 h, samples of the media were collected for progesterone RIA (32), and [3H]thymidine (3 µCi; Amersham, Amersham Intl. plc., Buckinghamshire, UK) was added to the cell cultures before measurement of DNA synthesis (see below), and the incubation was continued until 24 h.

Measurement of DNA Synthesis
A method described previously (33) was used with some modifications. The cells were fixed by incubation for 10 min in 0.5 ml of ice-cold 100% MeOH. The fixed cells were washed once with 1x HBSS (GIBCO) and once with 1 ml of ice-cold 10% trichloroacetic acid, incubated for 10 min, and washed again twice with 10% trichloroacetic acid for 5 min. After addition of 0.5 ml of 0.3 N NaOH/1% sodium dodecyl sulfonate, the cell were incubated for 30 min at room temperature. The tritium content in the cell lysates was determined by liquid scintillation spectrometry.

LH Receptor-Binding Assay
Highly purified hCG (CR-121) was iodinated with [125I]NaI (IMS 300, Amersham) using a solid-phase lactoperoxidase method described previously (34, 35). The specific activity of the preparation used was 50 Ci/g, with maximum binding of 25% to an excess of mouse testicular membranes. For Scatchard analysis, the C{alpha}1 adrenal tumor cells (40,000 cells per tube) were incubated in a reaction volume of 250 µl with different dilutions of the tracer (15,000–500,000 cpm/tube) and in the presence and absence of 50 IU of unlabeled hCG (Pregnyl; 3,000 IU/mg, Organon, Oss, The Netherlands). As a positive control, the LHR were determined as above in a mouse Leydig tumor cell line, BLT-1 (2).

For LHR assay of normal and tumorous adrenal tissues, pairs of the glands were homogenized in 2 ml of Dulbecco’s PBS + 0.1% BSA, and 100-µl aliquots of the crude homogenates were incubated with 100,000 cpm/tube of the [125I]iodo-hCG tracer, in the presence and absence of 50 IU of nonradioactive hCG (Pregnyl). The LH binding was corrected according to the protein content of the adrenal gland homogenates (36). hCG binding of the mouse testis homogenate, measured in the same way, was used as positive control.

Northern Hybridization
Total RNA from mouse testis, normal and tumorous adrenal tissue, and C{alpha}1 cells was isolated by the guanidinium isothiocyanate/CsCl2 method (37). Twenty micrograms of total RNA were resolved on 1.2% formaldehyde denaturing agarose gel and transferred onto nylon membrane (Hybond-N, Amersham). For hybridization, a complementary RNA probe for the rat LHR was generated from a fragment of the LHR cDNA, spanning nucleotides 441–849 of its extracellular domain, subcloned into pGEM-4Z plasmid (38). The 32P-labeled probe was generated using a Riboprobe system II kit (Promega, Madison, WI) and the cDNA as template. Prehybridization, hybridization, and subsequent membrane washings were performed as described previously (39). The filters were exposed to x-ray film (Kodak XAR-5, Eastman Kodak, Rochester, NY) at -70 C for up to 7 days.

Detection of LHR and FSH Receptor mRNA by RT-PCR
Total RNA from tissues and cell were extracted as described above (37). Two micrograms of total RNA were reverse-transcribed using the avian myeloma virus RT (Promega, Madison, WI), and the primer pairs used were specific for the rat FSH and LH receptor sequences. For the FSH receptor, the sense primer, 5'-ATGGCTGAGTAAGAATGGGA-3', corresponded to nucleotides 560–579, and the antisense primer, 5'-CTTGCCTTAAAATAGACTTGTTGC-3', corresponded to nucleotides 933–908 of the cDNA (40, 41). For the LH receptor, the sense primer, 5'-CTCTCACCTATCTCCCTGTC-3', corresponded to nucleotides 179–195, and the antisense primer, 5'-TCTTTCTTCGGCAAATTCCTG-3', corresponded to nucleotides 878–858 of the respective cDNA (39, 42). For amplification, the Dynazyme thermostable recombinant DNA polymerase (Promega, Madison, WI) was used in a thermal cycler. In the first step, reaction was started at 50 C for 10 min (RT), followed by a period of 3 min at 97 C and then run for 40 PCR cycles (96 C for 1.30 min; 53 C for 1.30 min; 72 C for 2 min) and the final extension for 10 min at 72 C, as described by us previously for LH receptor and FSH receptor cDNA amplification (39, 42).

Immunocytochemistry
Paraformaldehyde-fixed paraffin sections (5 µm thick) of adrenal glands were dewaxed, incubated in 0.3% H2O2 in ethanol to block endogenous peroxidases, rehydrated, and incubated further with 3% normal goat serum in Tris-buffered saline (pH 7.6). Serial sections of the adrenal glands were reacted either with a polyclonal rabbit antiserum directed against the N-terminal sequence (amino acids 15–38) of the rat LHR (1:250–1:1000 dilution of the lyophilysate in H2O) (kindly donated by Dr. P. C. Roche, Rochester, MN) or a rabbit polyclonal anti-Tag antibody (1:500–1:5000 dilution in PBS) (kindly donated by Dr. D. Hanahan, University of California, San Francisco, CA) (43). The slides were incubated at 4 C overnight. The bound antibodies were visualized with the immunoperoxidase technique (Vectastain Elite ABC kit, Vector, Burlingame, CA).

Statistical Analysis
A MacIntosh version of the SuperANOVA program (Abacus Concepts, Inc., Berkeley, CA) was used for one-factor ANOVA, followed by factorial tests, Duncan’s new multiple range test, and Fisher’s protected least significant difference post hoc tests.


    ACKNOWLEDGMENTS
 
We thank Ms. Maritta Forsblom and Ms. Jenni Laine for their excellent care of the mice and Ms. Riikka Kytömaa and Ms. Tarja Laiho for skillful technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Professor Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi

This work was supported by grants from The Sigrid Jusélius Foundation, The Finnish Cancer Societies, and The Academy of Finland.

Received for publication November 3, 1997. Revision received January 23, 1998. Accepted for publication February 13, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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