An Activated Human Follicle-Stimulating Hormone (FSH) Receptor Stimulates FSH-Like Activity in Gonadotropin-Deficient Transgenic Mice

Miriam Haywood, Nina Tymchenko, Jenny Spaliviero, Adam Koch, Mark Jimenez, Jörg Gromoll, Manuela Simoni, Verena Nordhoff, David J. Handelsman and Charles M. Allan

Andrology Laboratory (M.H., N.T., J.S., A.K., M.J., D.J.H., C.M.A.), ANZAC Research Institute, Sydney, New South Wales 2139, Australia; and Institute of Reproductive Medicine (J.G., M.S., V.N.), University of Münster, D-48419 Münster, Germany

Address all correspondence and requests for reprints to: Dr. Charles M. Allan, Andrology Laboratory, ANZAC Research Institute, Sydney, New South Wales 2139, Australia. E-mail: charles{at}med.usyd.edu.au.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR+) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR+, the rat androgen-binding protein gene promoter was used to direct FSHR+ transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR+ mRNA. Testis weights of transgenic FSHR+ hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR+-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal (~2-fold) and FSH-stimulated (~50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR+, respectively. Transgenic FSHR+ also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR+ response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR+ activity independent of androgen-specific actions. The FSHR+ response was male specific as ovarian expression of FSHR+ had no effect on hpg ovary size. These findings reveal transgenic FSHR+ stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IT IS WELL established that the pituitary-derived FSH plays an important role in testicular development and spermatogenesis. The testicular actions of FSH are mediated via a specific G protein-coupled cell surface receptor uniquely expressed in Sertoli cells (1). Ligand-dependent activation of the FSH receptor (FSHR) initiates the production of intracellular cAMP (2) and Ca2+ (3) second messengers, which activate signal transduction pathways responsible for the perinatal proliferation of Sertoli cells and their governing of postnatal maturation of germ cell populations (4, 5). The precise downstream molecular pathways selectively activated by the FSHR have yet to be clearly established.

In humans, the absolute requirement for FSH in male fertility remains uncertain (6, 7, 8). While the prevailing view is that fertility in man requires both androgen and FSH actions, previous studies have identified a mutation in the FSHR gene that inactivates the ligand-binding capacity of the mutated receptor and subsequent cAMP response (9). The inactivated receptors completely disrupt female fertility but males remain fertile despite having smaller testes (6). Other inactivating FSHR mutations have been identified as causing reproductive disruption in females but not males (10). Whether these receptor mutations result in a complete block of FSH activity in vivo has yet to be confirmed. Conversely, a proposed activating mutation in the FSHR was identified in a hypophysectomized man who retained spermatogenesis and fertility despite pituitary tumor-related gonadotropin deficiency (11). This mutant receptor contained a single amino acid substitution (Asp567Gly) in the third cytoplasmic loop that corresponded to similar sites of activating mutations in related LH (12), and TSH receptors (13). It was proposed that this mutation resulted in the only known constitutively active FSHR+ identified in humans to date, which may have induced the necessary FSH bioactivity independent of ligand in this FSH-deficient man. More recent studies demonstrated that FSHR+ increased basal levels of cAMP production in a transfected Sertoli cell line (1), whereas other studies using nontesticular cell lines have produced conflicting results regarding the in vitro bioactivity of this activating mutation (14, 15). Apart from the original patient expressing this mutated FSHR+, the specific biological actions of this human FSHR+ in vivo have yet to be confirmed.

We have previously used the hypogonadal (hpg) mouse (16) as a model of congenital and functionally complete gonadotropin deficiency to investigate the role of FSH or androgens in testicular development, in particular spermatogenesis (17, 18, 19, 20). The gonads of hpg mice fail to develop postnatally due to a major deletion in the GnRH gene (21) but remain functionally responsive to exogenous androgen and FSH treatment (17, 18). This constitutes a unique null reproductive hormone background on which to examine selective hormonal effects on postnatal testicular function. Using this approach, we recently created a novel transgenic (Tg) hpg paradigm to investigate the specific gonadal actions of FSH (20). Tg-FSH expression on the hpg background stimulated Sertoli cell maturation, and spermatogenesis progressed to sparse numbers of postmeiotic germ cells at the elongated spermatid stage. This testicular phenotype was similar to observations in complementary mouse models with preserved FSH expression and selective LH (and therefore androgen) deficiency due to targeted disruption of the LH receptor gene (22, 23). Therefore, the gonadotropin-deficient background of the hpg mouse provides a valuable paradigm to critically evaluate FSH-dependent actions in the testis and provides a further advantage with the ability to examine FSHR activity in isolation or in combination with androgen actions.

We have now created a novel Tg-hpg model to selectively study the actions of this mutated FSHR+ expressed in the testis. This unique Tg approach represents the first in vivo paradigm reported to date that examines the actions of a Tg gonadotropin receptor expressed independently of its cognate ligand. Our initial analysis of Tg-hpg males demonstrated that expression of human FSHR+ in vivo resulted in autonomous FSH-like activity in the mouse testis despite the absence of its circulating ligand.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of Tg-FSHR+
Tg animals were prepared using the rat androgen binding protein (rABP)-FSHR+ DNA construct (Fig. 1AGo). The rABP promoter sequence directs Sertoli cell-specific transgene expression in the mouse testis (24). Two independent Tg lines (RR3, RR4) were used to express Tg-FSHR+ on the hpg background, which was previously shown to have no detectable circulating FSH (16, 19, 25). Hemizygous (+/-) Tg-hpg males were generated at a level (5.6% of offspring) close to the expected frequency (6.3%). Tg-FSHR+ mRNA was expressed in the testis and ovary of both normal (non-hpg) or hpg animals as determined by RT-PCR (Fig. 1BGo). Ovary FSHR+ expression was lower than the testis by semiquantitative PCR (data not shown). This assay showed no cross-reactivity to the endogenous mouse FSHR sequence, and deoxyribonuclease treatment of all RNA samples ensured that the PCR products detected were not due to contamination by genomic Tg-DNA, as confirmed by PCR amplification of RNA samples before reverse transcription (RT) treatment (no RT controls). Further analysis of Tg-FSHR+ mRNA expression in six different mouse tissues revealed that FSHR+ mRNA was most abundant in the testis, and also present in the brain and at lower levels in the pituitary, with no expression detected in the liver, kidney, or spleen (Fig. 1CGo). The expression of Tg-FSHR+ was further confirmed by [125I]FSH binding to testis homogenates. Testes from RR3 and RR4 Tg lines showed a comparable increase in the maximal specific binding of [125I]FSH (377 ± 25, n = 5; and 422 ± 39, n = 8 cpm/mg tissue, respectively) relative to non-Tg controls (211 ± 42, n = 5 cpm/mg tissue).



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Figure 1. Transgene FSHR+ Construct and mRNA Expression

A, The Tg-DNA construct consisted of the human FSHR+ minigene (exons indicated by Roman numerals) flanked by the rABP gene promoter P1 and polyadenylation signal (polyA) sequence. The FR1/FR2 primers and amplified RT-PCR product are shown below. B, Expression of human Tg-FSHR+ mRNA was determined by RT-PCR analysis using total testis or ovary RNA from age-matched Tg-FSHR+ and non-Tg mice of the hpg (GnRH gene -) or normal (GnRH gene +) genotype. Amplified DNA fragments were visualized by ethidium bromide staining after agarose gel electrophoresis. Lanes were loaded with either control PCR of Tg plasmid DNA (lanes 1 and 6), RT-PCR of testis RNA (lanes 2, 4, 7, 9, and 15) or no-RT (reverse transcription) control PCR of testis RNA to rule out Tg-DNA contamination (lanes 3, 5, 8, and 10), and RT-PCR of ovary RNA (11 12 13 14 ). There was no amplification of the endogenous mouse FSHR sequence, and actin mRNA was detected as an internal control. C, Expression of Tg-FSHR+ mRNA expression in six different mouse tissues by RT-PCR. T, Testis; L, liver; B, brain; P, pituitary; S, spleen; K, kidney. Mouse actin mRNA was detected as internal control for each tissue.

 
In Vitro Bioactivity of Tg-FSHR+ in Cultured Sertoli Cells
Sertoli cells were isolated from immature Tg and non-Tg littermates to examine the levels of second messenger cAMP produced in the presence of mutant FSHR+. Intracellular cAMP levels in the absence of FSH stimulation were significantly increased 1.8-fold in cultured Tg Sertoli cells compared with non-Tg cells (Fig. 2Go). The basal cAMP levels of FSHR+ Sertoli cells were also increased to similar levels (1.5- to 2.0-fold) in two other independent Tg lines examined (data not shown), confirming that the expression of Tg-FSHR+ produced a ligand-independent rise in cAMP production. To verify cell-surface expression of human Tg-FSHR+, the Sertoli cells were stimulated with recombinant human (rh)FSH. The cAMP response (at plateau) to maximal FSH treatment was 1.5-fold greater in Tg-FSHR+ Sertoli cells compared with control Sertoli cells (Fig. 2Go).



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Figure 2. Intracellular cAMP Levels in Cultured Primary Sertoli Cells

Sertoli cells were isolated from Tg-FSHR+ (line RR.3) or non-Tg littermates. Intracellular cAMP levels were determined in the presence of 0.5 mM 3-isobutyl-1-methylxanthine and corrected for total protein content of each sample. A, Basal levels of cAMP in the absence of FSH stimulation were significantly increased in Tg Sertoli cells compared with non-Tg cells (P < 0.03). B, The FSH-stimulated cAMP response in isolated Tg-FSHR+ (solid circles) or non-Tg (open circles) Sertoli cells. Intracellular cAMP levels were significantly higher in Tg-FSHR+ cells at doses of rhFSH greater than 100 ng/ml.

 
In Vivo Bioactivity of Tg-FSHR+
The gonadal expression of Tg-FSHR+ in normal (non-hpg) male or female mice did not disrupt normal reproductive function, as the average litter sizes from Tg hemizygous (+/-) males (7.7 ± 0.3, n = 32) or females (8.0 ± 0.6, n = 16) were equivalent to the litter size from non-Tg breeders (7.4 ± 0.2, n = 64). In Tg-FSHR+ gonadotropin-deficient hpg males the in vivo actions of FSHR+ were initially determined by comparison of testis weights with those of non-Tg hpg littermates. Tg-FSHR+ hpg testis weights of the two independent lines examined (RR.3, RR.4) were increased up to 5-fold, with a significant average increase of approximately 2-fold relative to non-Tg controls (Fig. 3Go). We further examined the gonadal actions of Tg-FSHR+ expression when combined with a testosterone (T) treatment (1-cm implant) corresponding to a maximal androgen response in the hpg testis (17). The testes of T-treated FSHR+ hpg mice were significantly larger than the maximal hpg testis response to T alone (Fig. 3Go), suggesting mutant FSHR+ expression produced an independent synergistic effect with androgen on testicular development. In contrast, the ovarian weights of FSHR+ hpg females (0.66 ± 0.90 mg, n = 11) were not significantly different compared with non-Tg-hpg controls (0.80 ± 0.11 mg, n = 17), despite the ovary expression of FSHR+ mRNA (Fig. 1Go). Similarly, the testes of 9- to 14-wk-old normal (non-hpg) Tg mice (204.0 ± 10.1 mg, n = 6) showed no difference in weight compared with non-Tg normal testes (204.6 ± 14.2 mg, n = 6).



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Figure 3. Testis Weights of hpg Mice

Average testis weights of 9- to 10-wk-old Tg-FSHR+ hpg males or non-Tg hpg littermates either untreated (n = 11 Tg and 13 non-Tg) or treated (n = 7 Tg and 14 non-Tg) with 1-cm T-filled SILASTIC implants (T), which were previously found to induce maximal levels of androgen-specific testis growth (17 ). Untreated hpg or T-treated Tg testes were significantly heavier than non-Tg controls (P < 0.02 and P < 0.001, respectively).

 
Effect of Tg-FSHR+ on Testicular Histology
The testes of non-Tg-hpg controls exhibit seminiferous tubules with immature Sertoli cells, no tubular lumens, and an undeveloped germinal epithelium layer with spermatogenesis blocked at the pachytene stage of meiosis when compared with normal testes (Fig. 4Go, A and B). In comparison, the enlarged testes of FSHR+ hpg mice contained round spermatids, as well as sparse numbers of elongated spermatids (Fig. 4Go, C and D). Furthermore, the Sertoli cells in FSHR+ hpg testes contained the tripartite nucleus structure associated with Sertoli cell maturation (26). These intratubular cell types observed in Tg-FSHR+ hpg testes were equivalent to those found in hpg animals expressing the human FSH heterodimer (20), with both exhibiting a low level of postmeiotic germ cell development as shown by the appearance of round and elongated spermatids (Fig. 4Go, D and E). Preliminary analysis of DNA ploidy by flow cytometry has confirmed the presence of haploid postmeiotic cells in Tg-FSHR+ testes (data not shown). The only phenotypic difference observed between the Tg-FSH and Tg-FSHR+ models was the increased numbers of lumina in the seminiferous tubules of the larger FSHR+ hpg testes, which were not observed in age-matched Tg-FSH hpg testes of equivalent weight (Fig. 4Go, C and E).



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Figure 4. Testicular Histology of Tg-FSHR+ Mice

Shown are toluidine blue-stained 3-µm sections of testes from 9-wk-old non-Tg hpg (A), non-Tg normal (B), Tg-FSHR+ hpg (C and D), or Tg-FSH hpg (E and F) males. The Tg-FSH and Tg-FSHR+ hpg testes used in panels C and D and E and F weighed 10.6 and 10.3 mg, respectively, compared with the average non-hpg testis of 2.5 mg. Scale bars represent 50 µm in all panels, and L indicates a lumen. The postmeiotic round or elongated spermatids are indicated by the triangle or longer narrow arrows, respectively (panels D and F), which were sparsely distributed in the testes of Tg-hpg males.

 
Effect of Tg-FSHR+ on Serum Gonadotropins
The circulating levels of mouse FSH and LH were examined to determine whether tissue-expression of FSHR+ had increased or altered gonadotropin levels in hpg mice. The levels of serum LH (<0.09 ng/ml, n = 5) and FSH (<0.7 ng/ml, n = 5) in FSHR+ hpg animals remained undetectable and equivalent to the hypogonadotrophic background of non-Tg-hpg mice. Serum FSH remained undetectable in T-treated FSHR+ hpg males (<0.7 ng/ml, n = 5).

Effect of Tg-FSHR+ on T Production
Serum and intratesticular T levels in 9- to 10-wk-old hpg males were measured to determine whether Tg-FSHR+ expression alone affected androgen production. The slight rise detected in serum T levels of Tg-FSHR+ hpg males was not significant, although the small increase in intratesticular T levels was significant compared with non-Tg-hpg males (Table 1Go). However, there was no correlation between the testis T levels and the size of the corresponding Tg-FSHR+ hpg testis (data not shown). To examine the possibility of unexpected and functional Tg-FSHR+ expression in testicular Leydig cells, which could potentially activate steroidogenesis constitutively, we examined T production by cultured Leydig cells isolated from FSHR+ or non-Tg testes. Human FSH did not stimulate T production by either Leydig cell preparation (Fig. 5Go), suggesting that there was no functional human Tg-FSHR+ expression on the cell surface of Leydig cells. The levels of LH-stimulated T production were equivalent for both Leydig cell preparations. In addition, there was no significant difference between basal levels of T production in either Tg or non-Tg Leydig cells (Fig. 5Go), further supporting the absence of direct Tg-FSHR+ constitutive activity in these steroidogenic cells.


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Table 1. Intratesticular and Serum T Levels of 9- to 10-wk-old hpg or Normal (Non-hpg) Tg-FSHR+ and Control Males

 


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Figure 5. T Production by Leydig Cells

Leydig cells were prepared from 6- to 8-wk-old mice and cultured as described in Materials and Methods. The bars represent the mean ± SEM (n = 4) for T levels in the absence or presence of rhFSH or human chorionic gonadotropin (hCG) for Tg-FSHR+ (dark bar) and non-Tg (white bar) Leydig cell preparations. There was no significant difference between the levels of T produced by cultured Leydig cells obtained from Tg-FSHR+ or non-Tg animals.

 
We also examined the possibility that any increase in serum androgen levels may be due to T arising from adrenal steroidogenesis with or without potential ectopic FSHR expression. The serum T levels in castrated Tg-FSHR+ (1.39 ± 0.20 nM, n = 7) and non-Tg littermates (1.37 ± 0.17 nM, n = 7) were equivalent, suggesting that there was no significant extratesticular contribution to circulating T in the Tg-FSHR+ males.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have created a novel Tg paradigm to evaluate the specific ligand-independent actions of a mutated activating FSHR+ in testicular function. The transfer of Tg-FSHR+ expression onto the gonadotropin-deficient background provided by the hpg mouse has enabled selective analysis of potential FSHR activity on the gonads. This unique Tg approach has revealed that FSHR+ expression can stimulate an autonomous FSH-like Sertoli cell response in a testis deprived of its cognate ligand, FSH. Our current findings provide the first in vivo evidence of this activated FSHR, the nature of which was inferred from the unique clinical background and in vitro studies of FSHR function using cultured Sertoli cell lines.

One important feature of Tg-FSHR+ expression was that it demonstrated no obvious phenotype on the normal mouse background. The gonadotropin-deficient hpg background was essential to observe the postnatal gonadal activity of FSHR+. In addition, Tg-FSHR+ did not produce supraphysiological effects. Previous Tg models have demonstrated that excessive FSH activity orders of magnitude above physiological levels can lead to abnormal gonadal hyperplasia, enhanced serum T levels, and infertility (27, 28). Pharmacological doses of FSH also produced testicular hypertrophy in rats (29). These FSH effects were consistent with an earlier report of several human patients with pituitary macroadenomas hypersecreting FSH that were also found to have testicular enlargement (30). In our present study, Tg-FSHR+ expression did not disrupt normal male reproductive function or testicular development, suggesting that unlike reported supraphysiological FSH models the effects of FSHR+ expression were well within, or did not exceed, a normal physiological FSH-like response. Furthermore, the actions of FSHR+ were restricted to the testis since there was no detectable difference in the rudimentary ovaries of hpg mice despite the ovarian expression of Tg-FSHR+ mRNA. The mechanism for a male-specific FSHR+ effect is unknown, but it resembles the male-limited characteristic of activated LH receptors causing isosexual precocious puberty (12, 31). This male limitation may arise from the ovary’s requirement for conjoint activity of LH and FSH as embodied in the two-cell, two-gonadotropin hypothesis of ovary function (32). The absent ovary effect may also reflect lower ovarian levels of Tg-FSHR+ mRNA expression directed by the rABP promoter, compared with the testis, which is consistent with the lower ovary ABP expression reported in Tg-ABP mice using this promoter (33).

These findings demonstrate the 1.4-kb rABP P1 promoter contains the necessary elements to direct both ovary and testis expression of heterologous transgenes. We further demonstrated Tg-FSHR+ mRNA expression in the brain and lower levels in the pituitary, but no detectable expression in the liver, spleen, or kidney. Therefore, our results challenge the initial report describing testis-specific expression of the rABP P1 promoter (34) but support the more recently described brain expression of Tg-ABP using the P1 promoter (33). The expression of Tg-FSHR+ in the brain or pituitary had no effect on the serum FSH or LH levels in FSHR+ hpg mice, which remained undetectable like the non-Tg hpg controls, thus providing strong evidence that the gonadal effects in Tg males were due to the local testicular expression of Tg-FSHR+.

In our current hpg model, expression of human Tg-FSHR+ initiated Sertoli cell maturation and a small degree of postmeiotic haploid germ cell formation with the appearance of sparsely distributed elongating spermatids. This cellular development was equivalent to that observed in hpg mice selectively expressing bioactive human FSH heterodimers in the absence of LH (20) or in mice selectively lacking the LH receptor (22, 23). Thus, our unique Tg approach has demonstrated that the FSHR+ response can initiate autonomous FSH-like actions in a gonadotropin-deficient background. However, the incomplete germ cell development observed in FSHR+ hpg testes contrasts with the original hypophysectomized male carrier of this mutant FSHR+, who sustained fertility in the absence of both gonadotropins (11). Spermatogenesis was established in this patient before hypophysectomy, after which the presence of the mutated FSHR+ was proposed to have maintained functionally complete spermatogenesis without detectable levels of FSH. The low level of postmeiotic germ cell development in FSHR+ hpg testes may indicate a difference in the capacity of the FSH-like response to initiate complete germ cell maturation, compared with its maintenance of spermatogenesis. A related caveat is that the undescended state of untreated hpg testes (regardless of Tg genotype) may lead to an underestimation of postmeiotic Tg-FSHR+ actions, if this incomplete descent is itself deleterious to testis development. The germ and Sertoli cell development observed in FSHR+ hpg testes did not display the histopathological features of cryptorchidism (35), and adult T-treated hpg testes develop qualitatively complete spermatogenesis of normal appearance at any age, suggesting there is no progressive effects of incomplete descent on the developmental potential of the testis, as would be expected for true cryptorchidism. Expression of Tg-FSHR+ will provide a unique paradigm to examine the effects of FSHR+ on the maintenance of spermatogenesis after its induction. A more subtle intratesticular difference was also observed between the Tg-FSH and Tg-FSHR+ models, with an increased number of lumina formed in the seminiferous tubules of larger FSHR+ hpg testes, which were not observed in age-matched Tg-FSH hpg testes of equivalent weight. It is possible the autonomous FSHR+ response initiated an increased level of tubular fluid secretion compared with Tg-FSH, although the underlying mechanism for this difference is not clear.

Our analysis of cultured Sertoli cells further indicated that the ligand-independent FSHR+ response is likely to involve constitutive induction of cAMP-dependent pathways. We consistently observed a 1.5- to 2-fold increase in basal cAMP production in primary Sertoli cells expressing Tg-FSHR+, which was similar to the reported 3-fold rise in basal cAMP content of a Sertoli cell line expressing FSHR+ (1). The cAMP response in FSH-stimulated primary Tg-FSHR+ Sertoli cells was higher than the corresponding cAMP response in non-Tg control Sertoli cells at higher doses of FSH. However, low FSH levels did not produce a different cAMP response in Tg and non-Tg cells. Since circulating FSH was undetectable in Tg or non-Tg hpg animals, this finding suggests any residual FSH, if present, in hpg males would be unlikely to account for different cAMP responses in Tg or non-Tg testes. Taken together, these findings confirm the cell-surface expression of Tg-FSHR+ in Sertoli cells and demonstrate increased constitutive induction of the second messenger cAMP in the absence of its ligand. It is noteworthy that another laboratory found that FSHR+ had no effect on basal cAMP levels in transfected embryonic kidney 293 cells, and despite an observed 4-fold increase in receptor affinity for FSH, a saturating dose of FSH on this mutated FSHR resulted in a significantly reduced cAMP effect (14). The mechanism responsible for the different cAMP responses after FSHR+ expression in Sertoli cells or 293 cells remains to be determined. It is possible that the nontesticular 293 cells may be lacking Sertoli cell-specific components required for the second messenger response of the FSHR+. In our current model, the increased specific binding of [125I]FSH to Tg testes suggests increased affinity or more receptor sites; however, such changes are unlikely to account for higher cAMP levels in the absence of serum FSH in the hpg mouse. Previous in vitro studies have showed that basal levels of cAMP are not increased by higher expression (increased transfection DNA), and therefore density of wild-type FSHR (1, 14), whereas only mutant FSHR+ elevated the basal cAMP response in Sertoli cells (1). Similar characteristics have been reported for other G protein-coupled receptors, such as the wild-type and activated mutant forms of glucagon (36) or PTH receptors (37). More recent studies using COS-7 cells also described an increased basal cAMP response using FSHR+, and although not significantly increased, the authors noted that these findings did not exclude some degree of constitutive activity when allowing for membrane expression of binding-competent receptors (15). In summary, these in vitro findings indicate the ligand (FSH)-independent increase in cAMP levels in Tg Sertoli cells supports the presence of an activated FSHR+.

The present findings demonstrated that the Sertoli cell pathways activated by the mutant FSHR+ have a synergistic effect on testicular development when combined with androgen treatment. Our previous work showed that T treatment with a 1-cm implant resulted in a maximal androgen-specific induction of spermatogenesis and testis size on the hpg background (17). Expression of Tg-FSHR+ further enhanced the response of hpg testes to this maximal T dose, which indicated the FSHR+ effects were not solely due to the small but significant increase in testicular androgen synthesis. While our findings showed FSHR+ expression stimulated androgen-independent actions, we cannot rule out the possibility that FSHR+ also enhanced the sensitivity of the testis to very low levels of circulating androgens present in hpg males. However, the low level of germ cell maturation beyond the androgen-sensitive postmeiotic stage suggested that an androgen-specific germ cell response, if any, was minimal. Moreover, there was no positive correlation between testicular T levels in the Tg-FSHR+ hpg males and testis size. Interestingly, the increased testicular T levels in FSHR+ hpg testes are consistent with the original hypophysectomized male in which this mutant FSHR was first identified, who exhibited higher than castrate levels of circulating T after androgen withdrawal (11). Therefore, we propose this activated receptor can promote a low degree of androgen synthesis in the absence of LH, in addition to the FSH-like Sertoli cell effect. This androgen response may represent a paracrine effect as it was not via direct FSHR+ expression on Leydig or adrenal cells, or altered serum LH levels. Serum T levels in castrated Tg or non-Tg males were equivalent, suggesting that the adrenal contribution to circulating T in the Tg-FSHR+ mice is negligible. Likewise, there was no significant difference between basal levels of T production by cultured Leydig cells derived from Tg-FSHR+ and non-Tg testes, or production levels after treatment with rh-FSH, demonstrating the absence of Tg-FSHR+ constitutive activity in steroidogenic cells.

In summary, our unique Tg model has demonstrated that the expression of the first reported activated mutant FSHR can stimulate a cAMP response in Sertoli cells and autonomous FSH-like actions in the gonad in a ligand-deficient environment. This novel Tg approach provides a valuable in vivo paradigm to further investigate the molecular pathways involved in other intratesticular cellular signaling pathways including hormone responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Experimental Animals
All animal procedures were approved by the University of Sydney and Central Area Health Services Animal ethics committee and performed in accordance with the National Health and Medical Research Council code of practice for the care and use of animals and the NSW Animal Research Act (1985).

Preparation of FSHR+ Transgene DNA Construct
The human FSHR+ transgene was prepared by standard cloning procedures using the pBSSK-vector (Stratagene, La Jolla, CA). The human FSHR+ minigene consisted of exon 1 sequence from the BglII site 5' of the start codon, a shortened intron 1 sequence lacking the midregion, and exons 2–10 with no intervening intronic sequences. The human FSHR+ coding sequence was flanked by an upstream 1.4-kb rABP gene P1 promoter (24) and downstream polyadenylation signal sequence obtained from pSG5 (Stratagene), as illustrated in Fig. 1Go. Nucleotide sequences were confirmed by automated Dye-deoxy sequencing. The rABP-FSHR+ DNA Tg construct was digested with SacII and KpnI to remove most vector sequences and purified by agarose gel electrophoresis for microinjection into mouse oocytes as described below.

Generation of Tg Animals
Tg animals were prepared as described (20), using fertilized oocytes from the hpg strain derived from C3H/HeH x 101/H F1 hybrids (21). The FSHR+ construct was microinjected at 2–3 ng/µl in 10 mM Tris-HCl, (pH 7.4), 0.1 mM EDTA buffer. Six founders were obtained and three (RR1, RR3, RR4) were expanded to independent Tg lines; Tg animals were identified by PCR analysis of genomic DNA using primers FR1 5'-CAGAACCTTCCCAACCTTCA-3' and FR2 5'-GCTTCCATGAGGACGACAAG-3' to amplify a 452-bp fragment spanning exons 4–9 (Fig. 1Go). Transgene expression in vivo was confirmed by RT-PCR analysis of total testis or ovary RNA using the FR1/FR2 primers. To avoid amplification of contaminating Tg DNA sequences, total RNA isolated from freshly removed tissues using Tri reagent (Sigma, St. Louis, MO) was treated with ribonuclease-free deoxyribonuclease I (Life Technologies, Inc., Gaithersburg, MD), which was heat inactivated before reverse-transcription using Superscript II RT (Life Technologies, Inc.) as recommended. The corresponding endogenous mouse sequence was not amplified using standard PCR conditions and an annealing temperature of 57 C. Mouse actin RNA was used as internal standard. Mice expressing Tg-FSHR+ on a gonadotropin-deficient hpg background were obtained by cross-breeding animals heterozygous for the GnRH gene deletion, determined by detection of wild-type and hpg PCR products after agarose gel electrophoresis (17). Animals were housed under controlled conditions (12 h light-dark cycle, 19–22 C) with ad libitum access to food and water.

Animal Treatments and Serum and Tissue Collection
For T treatment of hpg mice, anesthetized 21-d-old Tg or non-Tg littermates or age-matched males received a subdermal 1-cm SILASTIC implant containing crystalline T as described (17). After 6 wk of T treatment animals were anesthetized and killed by terminal cardiac exsanguination, and collected serum was stored at -20 C. Fixed tissues were collected and weighed after left ventricle perfusion of anesthetized mice with 30 ml heparinized (10 IU/ml) saline prewarmed to 37 C, followed by 30 ml Bouins fixative containing 2% glutaraldehyde, 2% paraformaldehyde, 0.1% picric acid, in 0.2 M sodium phosphate buffer (pH 7.4). After overnight fixation, testes were transferred to 70% ethanol and reweighed. Dehydrated tissues were embedded in hydroxymethylmethacrylate resin (Technovit 7100, Kulzer and Co, Friedrichsdorf, Germany) according to the manufacturer’s instructions. Tissue sections (3 µm) were cut using a Polycut S microtome (Reichert Jung, Nossloch, Germany) and stained with 0.5% toluidine blue (Amerecso, Solon, OH). Control hpg testes were obtained from non-Tg littermates or age-matched males. In the castration study, testes of 6-wk-old anesthetized males were surgically excised, and 1 wk later serum was collected after cardiac exsanguination.

Cultured Sertoli and Leydig Cells
Sertoli cells were removed from dissected testes of 11- to 14-d-old mice with minor modification to previously described methods (3, 38). Briefly, collagenase and trypsinization removed interstitial and peritubular cells, after which Sertoli cell aggregates were plated (48-well plates, Costar, Cambridge, MA) and cultured in serum-free MEM (Life Technologies, Inc.) supplemented with 5 ng/ml sodium selenate, 3 µg/ml cytosine arabinoside, 5 µg/ml human transferrin, and 1 nM hydrocortisone at 37 C in a humidified atmosphere of 5% CO2 in air. After 3 d remaining germ cells were removed by hypotonic treatment with 20 mM Tris buffer for 2 min (39). On d 4, medium was replaced with serum-free supplemented MEM containing a phosphodiesterase inhibitor (0.5 mM 3-isobutyl-1-methylxanthine), and cells were untreated or stimulated with rhFSH (Serono Laboratories, Inc., Aubonne, Switzerland) for 1 h at 37 C. The incubation was terminated by removal of medium and addition of 0.1 M HCl for 20 min at 4 C. The cell extracts were collected and stored at -80 C for cAMP analysis. To obtain Leydig cells, the testes of 6- to 8-wk-old mice were removed, minced with fine scissors, and suspended in medium 199 containing 2% FCS, 2.4 mg/ml NaHCO3, and 20 mM HEPES (pH 7.4). Cells were filtered through a nylon 2-mm2 mesh, incubated for 1 h at 34 C, and then filtered through a 100-µm mesh and pelleted at 200 x g for 10 min at room temperature. The cell preparation was resuspended at 1 x 104 Leydig cells/ml in medium 199 with or without human chorionic gonadotropin (Serono Laboratories, Inc.) or rhFSH. Cells were incubated for 3 h in a shaking water bath (34 C, 80 cycles/min), gassed 2–3 min with carbogen at 30-min intervals, and pelleted; cell-free medium was collected and stored at -80 C for T assay.

cAMP and Hormone Assays
The cAMP content of Sertoli cell extracts corresponding to intracellular levels was determined by RIA as described previously (40). Assays were performed in at least triplicate, and the cAMP levels for each sample were normalized for total protein mg levels determined by the Bradford method (41). Total T levels in serum, cultured Leydig cell medium samples, or homogenized testes were measured in at least duplicate by RIA as previously described (17). Serum levels of mouse LH were measured by RIA using reagents from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDKD) as recommended. Incubations used 100 µl of serum or mouse LH standard (AFP-5306A) with 100 µl antiserum (rLH S-11) for 24 h at 22 C, followed by 100 µl [125I]rLH (rLH I-9) for an additional 24 h. Samples were counted in duplicate and the LH detection limit was 70 pg/ml. Serum levels of mouse FSH were determined in duplicate by time-resolved immunofluorometric assay using monoclonal antibody 56A (kindly provided by Organon, Oss, The Netherlands) as described previously (42), with minor modifications. Mouse FSH standard (AFP-5308D, NIDDKD) or serum samples were diluted in horse serum. The FSH assay detection limit was 0.6 ng/ml.

Testicular FSH Binding Assay
The specific binding of [125I]FSH to testis membranes was determined as described (43). Freshly isolated testes from immature mice were weighed and then homogenized in 0.5 ml assay buffer (40 mM Tris-HCl, 5 mM MgSO4, 0.1% BSA) on ice. Homogenates were centrifuged at 11,000 x g for 20 min at 4 C and membrane pellets stored at -80 C. Thawed membrane samples were resuspended in 100 µl assay buffer (2–4 mg of tissue), and duplicate tubes were incubated with 50 µl of [125I]rFSH I-9 (10,000 cpm/tube) for 6 h at 35 C, with or without excess rat FSH (20 µg RP1-NIH). After addition of 1 ml assay buffer, the tubes were centrifuged at 11,000 x g for 3 min at 4 C and the supernatant discarded, and the pellets were washed again then counted. Maximal specific binding was determined by subtraction of nonspecific binding (excess unlabeled hormone) from total (without excess).

Data Analysis
Data were expressed as mean ± SEM. Statistically significant differences (P < 0.05) among the various treatment groups were determined by Students paired or unpaired t test.


    ACKNOWLEDGMENTS
 
We thank F. Thorn and L. Pekel for excellent technical assistance.


    FOOTNOTES
 
This work was supported in part by a University of Sydney U2000 Research Fellowship, a National Health and Medical Research Council grant (153855), and the German Research Foundation (FOR 197/3).

Abbreviations: FSHR, FSH receptor; hpg, hypogonadal; rABP, rat androgen binding protein; rhFSH, recombinant human FSH; T, testosterone; Tg, transgenic.

Received for publication January 23, 2002. Accepted for publication July 24, 2002.


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