Failure of Normal Leydig Cell Development in Follicle-Stimulating Hormone(FSH) Receptor-Deficient Mice, But Not FSHß-Deficient Mice: Role for C
Abstract|, http://www.100md.com
Previous studies have suggested that FSH may be involved in regulation of Leydig cell function. We have examined this directly using two mouse models with null mutations in either the FSH ß-subunit (FSHßKO mice) or the FSH receptor (FSHRKO mice). Circulating LH levels were normal in adult FSHßKO mice, but were significantly increased in FSHRKO mice. Intratesticular testosterone levels increased normally in FSHßKO mice from birth to adulthood, whereas testosterone levels in FSHRKO mice failed to increase normally after puberty and were significantly reduced in adult animals. This was associated with reduced levels of mRNA encoding cytochrome P450 side-chain cleavage, 3ß-hydroxysteroid dehydrogenase type VI, and steroidogenic acute regulatory protein in FSHRKO mice. Leydig cell number was normal in FSHßKO mice during development, but in FSHRKO mice Leydig cell number increased slowly after puberty and was significantly reduced in the adult animal. Transfection studies showed that the FSHR exhibits constitutive activity in the absence of agonist stimulation. The results indicate, therefore, that Sertoli cells regulate the development of Leydig cell number and that constitutive activity within the FSHR is sufficient to stimulate this process. The presence of the hormone itself is not required when circulating LH levels are adequate.
Introduction6i[}*r, 百拇医药
ANDROGEN PRODUCTION and fertility in the adult male are dependent upon Leydig cell activity in the testis. These cells, in turn, depend crucially on LH secreted from the pituitary in response to GnRH. This is clearly illustrated by the failure of postnatal androgen production in mice lacking either GnRH peptide or the LH receptor (1, 2, 3). There is, in addition, a body of evidence suggesting that Leydig cell growth, activity, and survival are dependent upon the Sertoli cell population of the testis. This hypothesis dates back to earlier studies showing that FSH can act to increase Leydig cell activity in hypophysectomized rats or GnRH-deficient (hpg) mice (4, 5, 6, 7, 8, 9, 10, 11). As FSH receptors (FSHR) are present only in the Sertoli cells in the testis, this has been taken as evidence of Sertoli cell regulation of Leydig cell function. These earlier studies on the normal role of FSH in regulating the Leydig cell population were complicated by two issues. Firstly, circulating LH was markedly reduced or absent in the models used, and the Leydig cells were lacking any normal trophic stimulation. Secondly, in most cases [with two exceptions (9, 11)] the studies predated the introduction of recombinant FSH, and the FSH preparations used contained low, contaminating levels of LH. It is clear from more recent studies, in which Sertoli cell apoptosis was shown to be followed by Leydig cell degeneration (12), that there is a clear link between the two cell types, but the normal role of FSH in regulating Leydig cell development and activity remains unclear.
The recent generation of different models of FSH deficiency in the mouse now allows us to address this issue of FSH involvement in Leydig cell development and function. Mice lacking FSH through a null mutation in the ß-subunit (FSHßKO mice) were first described by Kumar et al. (13) and were shown to be fertile, but with reduced testis size. Later, two separate laboratories described the development of an FSHR-null (FSHRKO) mouse that is also fertile with reduced testis size (14, 15). Interestingly, circulating testosterone levels are reported to be normal in adult FSHßKO mice (13), but to be reduced in FSHRKO mice (14). In this study we examined Leydig cell development and function in both models directly. The results show that Leydig cell number and function appear normal in FSHßKO mice, but that Leydig cell number and, hence, androgen production are reduced in FSHRKO mice. Further studies indicate that this difference may be due to the presence of constitutive activity within the FSHR in the absence of hormone ligand.
Materials and Methods/96x[rx, 百拇医药
Animals/96x[rx, 百拇医药
Generation of both FSHßKO and FSHRKO mice has been described previously (13, 15). Both colonies were raised on a C57BL6 background and were maintained at University of Oxford under United Kingdom Home Office regulations. As both FSHRKO and FSHßKO adult males are fertile, the colonies were maintained by breeding homozygous males with heterozygous females. Heterozygous males were used as the control group in this study. Animals were killed at 1, 5, 20, 60, 120, and 180 d of age (day of birth is d 1). One testis from each animal was frozen in liquid N2, and the other testis was fixed overnight in Bouin’s fluid before being stored in 70% ethanol. Blood was also collected from adult animals, and serum was stored frozen at -20 C before measurement of LH. To measure the responsiveness of testes in vivo to exogenous stimulation, adult animals (60 and 180 d old) were injected ip with human chorionic gonadotropin (hCG; 0.5 IU/g; Serono Pharmaceuticals Ltd., Feltham, UK) in saline. Animals were killed 1 h after injection, and testes were stored frozen at -20 C until extracted for hormone assay.
Hormone and second messenger assays3su%:), http://www.100md.com
To measure intratesticular testosterone levels steroids were extracted from the testes in ethanol and measured by RIA as previously described (16). Levels of progesterone in culture medium were measured directly by RIA (17). Serum levels of LH were measured using an in-house immunofluorometric assay (Delfia, Wallac, Inc., Turku, Finland) as previously described (18). Levels of cAMP in culture medium were measured by RIA (19).3su%:), http://www.100md.com
Stereology and histology3su%:), http://www.100md.com
For stereology, testes were fixed in Bouin’s solution and stored in 70% ethanol. Tissue was embedded in Technovit 7100 resin (TAAB Laboratories Ltd., Reading, UK), cut into sections (20-µm thickness), and stained with Harris’ hematoxylin. The total testis volume was estimated using the Cavalieri principle (20), and the slides used to estimate the number of cells were also used to estimate testis volume. The optical disector technique (21) was used to count the number of Leydig cells in each testis. The numerical density of Leydig cells was estimated using an Olympus BX50 microscope fitted with a motorized stage (Prior Scientific Instruments, Cambridge, UK) and Stereologer software (Systems Planning Analysis, Alexandria, VA).
To generate semithin (1-µm) sections, testes were fixed in 1% glutaraldehyde-4% paraformaldehyde in phosphate buffer (0.1 M), pH 7.2, for 24 h at 4 C and embedded in araldite. Cut sections were stained with toluidine blue.n)#q2de, http://www.100md.com
Measurement of mRNA levelsn)#q2de, http://www.100md.com
Real-time PCR was used to measure levels of specific mRNA species present in the testes of control, FSHRKO, and FSHßKO animals (22). To allow results to be compared directly on a per testis basis, a standard amount of external control mRNA (luciferase) was added to each sample during the RNA extraction phase (22, 23). Sequences of primers and probes and the reaction conditions used were previously described (23).n)#q2de, http://www.100md.com
Cell transfectionsn)#q2de, http://www.100md.com
A mouse Leydig tumor cell line, mLTC-1 (24), was cultured in HEPES-buffered Waymouth’s medium (Sigma, St. Louis, MO) supplemented with 9% heat-inactivated horse serum (Life Technologies, Inc., Paisley, UK), 4.5% heat-inactivated fetal calf serum (BioClear, Calne, UK), and 50 mg/liter gentamicin (Biological Industries, Hemeek, Israel) at 37 C in 5% CO2. Cells were transiently transfected with an expression vector containing the mouse FSHR (pSG5-mFSHR-WT), the mouse FSHR with an inactivating point mutation (pSG5-mFSHR-C566T) (25, 26), or vector alone (pSG5) using Lipofectamine reagent (Life Technologies, Inc., Paisley, UK). Transfection efficiencies were verified by cotransfections with a luciferase expression vector. For transfections, cells were cultured on 10-cm diameter tissue culture plates to confluence of 50–70%, and 9 µg pSG construct vector or 0.3–0.6 µg luciferase vector were used to transfect the cells in Opti-MEM I with Glutamax (Invitrogen, Paisley, UK). After 5 h an equal volume of Waymouth’s medium supplemented with twice the normal concentration of serum was added to the cells. One day after the transfection cells were trypsinized and counted, and 60,000 cells/well were plated on 24-well plates. After 24 h cells were washed, and 1 ml Opti-MEM I with Glutamax plus 100 nmol/liter 1-methyl-3-isobutylxanthine (Alcon Laboratories, Inc., Milwaukee, WI) were added per well. Cells were incubated at 37 C in quadruplicate under basal conditions or in the presence of recombinant human FSH (National Hormone and Pituitary Program, McKesson BioServices, Rockville, MD). Aliquots of the incubation medium were collected after 2 h for cAMP and after 6 h for progesterone measurements.
Statistical analysis;, http://www.100md.com
Differences between control, FSHRKO, and FSHßKO animals were initially analyzed by single or two-factor ANOVA. Where a significant overall difference between animal groups was detected, differences between individual means were assessed by the Newman-Keuls test (after single factor analysis) or by t tests using the pooled variance (after two-factor analysis).;, http://www.100md.com
Results;, http://www.100md.com
Intratesticular testosterone levels;, http://www.100md.com
In control animals from both FSHßKO and FSHRKO groups testosterone levels per testis were low from birth to d 20, then increased markedly to d 60 and continued to rise up to d 180 (Fig. 1). In both FSHßKO and FSHRKO animals testosterone levels were normal in the prepubertal period up to d 20. After d 20 testosterone levels increased normally in the FSHßKO group, and on d 180 there was no significant difference from control values. In contrast, testosterone levels in FSHRKO mice increased more slowly after d 20 and were significantly less than control values on d 180 (Fig. 1).
fig.ommitteed/8dg5z, 百拇医药
Figure 1. Intratesticular testosterone concentrations in control heterozygous animals (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) animals during postnatal development. The results show testosterone levels per whole testis and represent the mean ± SEM of 4–17 animals/group. *, Significant (P < 0.05) difference from age-matched controls./8dg5z, 百拇医药
To measure the responsiveness of testes from FSHßKO and FSHRKO groups to exogenous stimulation by hCG, adult animals were injected with hCG as described in Materials and Methods. After hCG treatment, there was an increase in intratesticular testosterone levels in all groups, but levels in both FSHßKO and FSHRKO animals were significantly reduced compared with control values (Fig. 2). No difference was found between the two knockout models in their response to hCG./8dg5z, 百拇医药
fig.ommitteed/8dg5z, 百拇医药
Figure 2. Intratesticular testosterone levels per testis in adult control heterozygous mice (+/-) and in adult FSHßKO (-/-) and FSHRKO (-/-) mice after hCG injection. Animals were injected with hCG (0.5 IU/g) and were killed 1 h later. The mean ± SEM of four to nine animals per group are shown. Both 60- and 180-d-old animals were included in each group, but there was no age-dependent difference in testicular testosterone content after hCG injection, and data from different aged animals were pooled. Groups with different letter superscripts are significantly (P < 0.05) different.
Circulating LH levels47v, http://www.100md.com
Levels of circulating LH in adult FSHßKO mice did not differ significantly from those in control animals (Fig. 3). In contrast, LH levels in FSHRKO mice were significantly increased compared with control values (Fig. 3).47v, http://www.100md.com
fig.ommitteed47v, http://www.100md.com
Figure 3. Circulating LH levels in adult control heterozygous mice (+/-) and in adult FSHßKO (-/-) and FSHRKO (-/-) mice. The mean ± SEM of 4–12 animals/group are shown. The animals used in this study were 60 d old. Groups with different letter superscripts are significantly (P < 0.05) different.47v, http://www.100md.com
Expression of Leydig cell mRNA levels47v, http://www.100md.com
Levels of mRNA encoding the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase type VI (3ßHSD VI), cytochrome P450 17{alpha} -hydroxylase (P450c17), and 17ßHSD III and the steriodogenic acute regulatory protein (StAR) were measured by real-time PCR. Levels of mRNA were measured relative to an externally added standard (luciferase) that allows direct comparison of mRNA levels between groups (22). For each mRNA species, mean levels in FSHRKO mice were lower than those in control animals, and this difference was significant for P450scc, 3ßHSD VI, and StAR (Fig. 4). In FSHßKO mice expression levels of the mRNA species measured were either normal (P450scc, 3ßHSD VI, and P450c17) or slightly, but significantly, increased (17ßHSD III and StAR).
fig.ommitteed@1h, http://www.100md.com
Figure 4. Measurement of Leydig cell-specific mRNA species by real-time PCR. The results show expression in control heterozygous mice (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) mice. RNA was extracted from whole testes of adult animals and reverse transcribed to generate cDNA. Levels of specific mRNA species were measured by real-time PCR and expressed relative to an external control (luciferase). As the same amount of luciferase mRNA was added to each sample, the results can be compared directly. Results show the mean ± SEM of four animals in each group. The animals used in this study were 180 d old. Groups with different letter superscripts are significantly (P < 0.05) different.@1h, http://www.100md.com
Testis size, Leydig cell number, and interstitial morphology@1h, http://www.100md.com
Testis size was reduced to about one third of normal in both FSHßKO and FSHRKO mice with the effect evident after d 5 (Fig. 5). Leydig cell number in both FSHßKO and FSHRKO mice was normal at birth and up to d 5 postnatally (Fig. 6). After d 5 Leydig cell number increased markedly in control animals, reaching a peak in adult animals. In FSHßKO mice Leydig cell development was normal after d 5, and numbers in the adult animal were not significantly different from control values. In contrast, Leydig cell numbers in FSHRKO mice failed to increase normally after d 5 and were significantly less than control values in the adult animal (Fig. 6). Despite differences in Leydig cell number interstitial cell morphology was similar in control, FSHßKO, and FSHRKO mice at the light microscopic level (Fig. 7.
fig.ommitteed*&f, 百拇医药
Figure 5. Changes in testicular volume during postnatal development in control heterozygous (+/-) mice and in FSHßKO (-/-) and FSHRKO (-/-) mice. Results show the mean ± SEM of three to five animals in each group.*&f, 百拇医药
fig.ommitteed*&f, 百拇医药
Figure 6. Leydig cell number during development in control heterozygous mice (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) mice. Leydig cell number was measured using the optical dissector method. Results show the mean ± SEM of three to five animals in each group. *, Significant (P < 0.05) difference from age-matched controls.*&f, 百拇医药
fig.ommitteed*&f, 百拇医药
Figure 7. Light micrographs showing interstitial tissue in control, FSHßKO, and FSHRKO mice. The animals used to prepare these micrographs were 60 d old. Bar, 10 µm. The control animal used in this montage was an FSHß heterozygote. There were no clear differences in the morphology of the interstitial tissue between the different groups of animals at the light microscopic level.
Constitutive FSHR activity1w1f7tq, 百拇医药
To determine whether the FSHR shows constitutive activity, a Leydig cell line was transfected with either wild-type FSHR (mFSH-WT) or control vector, and the activity of the cells was measured. Monitoring by luciferase coexpression showed that transfection efficiencies with the different plasmids were similar in the individual experiments. The results in Fig. 8A show data from a single experiment in which cAMP levels were measured after transfection, and it is clear that there was a significant increase in basal cAMP production in the presence of the receptor and the absence of ligand. In contrast, transfection with an FSHR containing an inactivating point mutation (mFSH-C566T) had no effect on basal expression. As expected, transfection with mFSHR also conferred sensitivity to FSH, which was significantly reduced in cells transfected with mFSH-C566T. Figure 8B shows the combined results from five experiments designed to measure changes in basal cAMP and progesterone production by Leydig cells after transfection with the FSHR. Overall, there was an approximately 4-fold increase in both measures of basal cell activity after transfection with the receptor.
fig.ommitteed!w)4m+, 百拇医药
Figure 8. Progesterone and cAMP production in a Leydig cell line transfected with mFSHR (mFSHR-WT), mFSHR with an inactivating point mutation (mFSHR-C566T), or vector only (control). A, Results from a single experiment showing cAMP production by transfected cells under basal conditions and in the presence of increasing levels of hFSH. Results show the mean ± SEM of quadruplicate wells. B, Combined data from five experiments showing basal cAMP and progesterone production in cells transfected with mFSHR-WT or vector alone (control). Results show the mean ± SEM.!w)4m+, 百拇医药
Discussion!w)4m+, 百拇医药
FSH is a member of the heterodimeric glycoprotein hormone family, which includes LH, hCG, and TSH. Members of this family share a common {alpha} -subunit with biological specificity conferred by a hormone-specific ß-subunit, although only the heterodimers show biological activity. As with other members of the family, FSH acts through a single receptor type although considerable variation in the receptor can be induced through alternate splicing (26, 27). It might be expected, therefore, that the effects of induced null mutations in the hormone ß-subunit gene or the hormone receptor gene would induce similar phenotypes in the affected animals. Consistent with this, it has been shown that both FSHßKO and FSHRKO male mice are fertile, but have reduced testis size and reduced levels of spermatogenesis (13, 14, 15). The results reported here, however, show that there are significant differences in the effects of the mutations on Leydig cell development in affected animals.
Leydig cell development appears to be normal in FSHßKO mice, with no difference in Leydig cell number during development or in the adult animal and no difference in testosterone or circulating LH levels. This confirms previous reports showing that Leydig cell number and circulating testosterone levels are normal in adult FSHßKO mice (28). In contrast, testosterone production by FSHRKO mice was reduced despite an increase in circulating LH, and this was associated with reduced testicular expression of key mRNA species associated with steroidogenesis in Leydig cells. It is likely, however, that these apparent changes in Leydig cell function are due to a failure of normal Leydig cell proliferation/differentiation during puberty in FSHRKO mice, causing the numbers of cells in the adult animal to be reduced to about 60% of normal. This would explain the reduction in levels of intratesticular testosterone and Leydig cell-specific mRNA species and the rise in circulating LH, which is inversely linked to overall changes in testosterone production. In a recent study of another FSHRKO mouse model, testosterone levels were also significantly reduced in the adult animals (29), and it is likely that this is due to the same mechanism of action.
When a more marked phenotype arises from a receptor null mutation compared with a hormone null mutation, it is likely that this is either because the receptor is not specific to the hormone targeted with the mutation or because the receptor itself shows constitutive activity in the absence of agonist stimulation. In the case of the FSHR, there is as yet no evidence that a growth factor or hormone, other than FSH, can activate the receptor. Previous studies have shown, however, that G protein-coupled receptors can display constitutive activity (30), and studies reported here show clearly that the FSHR will express constitutive activity in the absence of ligand. The FSHR normally shows down-regulation in the presence of FSH (31), and it would be expected, therefore, that receptor levels will be increased in FSHßKO mice. As constitutive activity varies directly with receptor number, this will serve to increase activation of the cells and provides a clear explanation for phenotypic differences between FSHRKO and FSHßKO mice. In this respect the FSH and TSH receptors appear to show similar ligand-independent activation, which differs from the LH receptor, which is reported only to show ligand-independent activity under nonphysiological conditions (32).
Two generations of Leydig cells arise during normal testis development: a fetal population, which appears shortly after testis differentiation in utero, and an adult population, which arises shortly before puberty (starting around d 5–10 in the mouse) (33, 34). In both FSHßKO and FSHRKO mice, Leydig cell number and testicular testosterone levels were normal on d 1 and 5, showing that FSH and the FSHR do not appear to play a role in the development of the fetal population. During fetal growth in utero, Leydig cell function develops normally in GnRH-deficient mice (1) and in mice with a null mutation in the common {alpha} -subunit (35), and it is not unexpected, therefore, that Leydig cell function is normal on the day of birth in FSHßKO and FSHRKO mice. Shortly after birth, fetal Leydig cells become critically dependent on gonadotropins (1), but results reported here show that neither FSH nor its receptor is required at this time.3ak.9, http://www.100md.com
Within the testis only the Sertoli cell population expresses the FSHR, and the effect of the FSHR null mutation on Leydig cell development must, therefore, be mediated through Sertoli cells. This implies that factors released by Sertoli cells act to induce normal Leydig cell proliferation/differentiation. From other studies of mutant mice two Sertoli cell-derived factors (desert hedgehog and platelet-derived growth factor A) have been implicated in this process (36, 37). It is likely that the FSHR is a permissive factor in this process, ensuring that overall Sertoli cell activity is high enough to maintain normal output of the trophic factors. It is clear, however, that FSH is required for normal levels of Sertoli cell activity, because in both FSHßKO and FSHRKO mice cell number is reduced, and spermatogenesis is compromised (28, 38).
Given that LH levels, Leydig cell number, and testosterone levels are normal in FSHßKO mice, it is not clear why intratesticular testosterone levels fail to increase normally after hCG stimulation. It may simply be due to a physical effect of the 50% reduction in size of the FSHßKO testis, which will affect blood flow to the testis interstitium and may limit the amount of testosterone that can actually be held within the testis. Alternatively, it is possible that the sensitivity of the testis to hCG stimulation or the maximum steroidogenic capacity of the Leydig cells is reduced in the absence of FSH. This does not, however, appear to affect androgen production under normal conditions.zp?c, http://www.100md.com
Previous studies showing an effect of FSH injections on Leydig cell activity were carried out using animals with low or absent circulating LH (4, 5, 6, 7, 8, 9, 10, 11). Results from this study suggest that in the presence of LH, FSH is not required for normal Leydig cell development as long as FSHR are present. The lack of effect of the FSHß null mutation on Leydig cell number contrasts with the almost complete failure of postpubertal development of Leydig cell number in GnRH-deficient mice, which lack LH and FSH (39). Thus, LH alone is sufficient for normal postnatal Leydig cell development (in the presence of the FSHR), but in the absence of LH, evidence from earlier studies suggests that FSH can induce stimulation of Leydig cell activity (9, 11).
Received June 20, 2002.q\n, 百拇医药
Accepted for publication September 13, 2002.q\n, 百拇医药
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Krishnamurthy H, Babu PS, Morales CR, Sairam MR 2001 Delay in sexual maturity of the follicle-stimulating hormone receptor knockout male mouse. Biol Reprod 65:522–531]@(6h4%, 百拇医药
Baker PJ, O’Shaughnessy PJ 2001 Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice. Reproduction 122:227–234(Paul J. Baker Pirjo Pakarinen Ilpo T. Huhtaniemi Margaret H. Abel Harry M. Charlton T. Rajendra Kuma)
Previous studies have suggested that FSH may be involved in regulation of Leydig cell function. We have examined this directly using two mouse models with null mutations in either the FSH ß-subunit (FSHßKO mice) or the FSH receptor (FSHRKO mice). Circulating LH levels were normal in adult FSHßKO mice, but were significantly increased in FSHRKO mice. Intratesticular testosterone levels increased normally in FSHßKO mice from birth to adulthood, whereas testosterone levels in FSHRKO mice failed to increase normally after puberty and were significantly reduced in adult animals. This was associated with reduced levels of mRNA encoding cytochrome P450 side-chain cleavage, 3ß-hydroxysteroid dehydrogenase type VI, and steroidogenic acute regulatory protein in FSHRKO mice. Leydig cell number was normal in FSHßKO mice during development, but in FSHRKO mice Leydig cell number increased slowly after puberty and was significantly reduced in the adult animal. Transfection studies showed that the FSHR exhibits constitutive activity in the absence of agonist stimulation. The results indicate, therefore, that Sertoli cells regulate the development of Leydig cell number and that constitutive activity within the FSHR is sufficient to stimulate this process. The presence of the hormone itself is not required when circulating LH levels are adequate.
Introduction6i[}*r, 百拇医药
ANDROGEN PRODUCTION and fertility in the adult male are dependent upon Leydig cell activity in the testis. These cells, in turn, depend crucially on LH secreted from the pituitary in response to GnRH. This is clearly illustrated by the failure of postnatal androgen production in mice lacking either GnRH peptide or the LH receptor (1, 2, 3). There is, in addition, a body of evidence suggesting that Leydig cell growth, activity, and survival are dependent upon the Sertoli cell population of the testis. This hypothesis dates back to earlier studies showing that FSH can act to increase Leydig cell activity in hypophysectomized rats or GnRH-deficient (hpg) mice (4, 5, 6, 7, 8, 9, 10, 11). As FSH receptors (FSHR) are present only in the Sertoli cells in the testis, this has been taken as evidence of Sertoli cell regulation of Leydig cell function. These earlier studies on the normal role of FSH in regulating the Leydig cell population were complicated by two issues. Firstly, circulating LH was markedly reduced or absent in the models used, and the Leydig cells were lacking any normal trophic stimulation. Secondly, in most cases [with two exceptions (9, 11)] the studies predated the introduction of recombinant FSH, and the FSH preparations used contained low, contaminating levels of LH. It is clear from more recent studies, in which Sertoli cell apoptosis was shown to be followed by Leydig cell degeneration (12), that there is a clear link between the two cell types, but the normal role of FSH in regulating Leydig cell development and activity remains unclear.
The recent generation of different models of FSH deficiency in the mouse now allows us to address this issue of FSH involvement in Leydig cell development and function. Mice lacking FSH through a null mutation in the ß-subunit (FSHßKO mice) were first described by Kumar et al. (13) and were shown to be fertile, but with reduced testis size. Later, two separate laboratories described the development of an FSHR-null (FSHRKO) mouse that is also fertile with reduced testis size (14, 15). Interestingly, circulating testosterone levels are reported to be normal in adult FSHßKO mice (13), but to be reduced in FSHRKO mice (14). In this study we examined Leydig cell development and function in both models directly. The results show that Leydig cell number and function appear normal in FSHßKO mice, but that Leydig cell number and, hence, androgen production are reduced in FSHRKO mice. Further studies indicate that this difference may be due to the presence of constitutive activity within the FSHR in the absence of hormone ligand.
Materials and Methods/96x[rx, 百拇医药
Animals/96x[rx, 百拇医药
Generation of both FSHßKO and FSHRKO mice has been described previously (13, 15). Both colonies were raised on a C57BL6 background and were maintained at University of Oxford under United Kingdom Home Office regulations. As both FSHRKO and FSHßKO adult males are fertile, the colonies were maintained by breeding homozygous males with heterozygous females. Heterozygous males were used as the control group in this study. Animals were killed at 1, 5, 20, 60, 120, and 180 d of age (day of birth is d 1). One testis from each animal was frozen in liquid N2, and the other testis was fixed overnight in Bouin’s fluid before being stored in 70% ethanol. Blood was also collected from adult animals, and serum was stored frozen at -20 C before measurement of LH. To measure the responsiveness of testes in vivo to exogenous stimulation, adult animals (60 and 180 d old) were injected ip with human chorionic gonadotropin (hCG; 0.5 IU/g; Serono Pharmaceuticals Ltd., Feltham, UK) in saline. Animals were killed 1 h after injection, and testes were stored frozen at -20 C until extracted for hormone assay.
Hormone and second messenger assays3su%:), http://www.100md.com
To measure intratesticular testosterone levels steroids were extracted from the testes in ethanol and measured by RIA as previously described (16). Levels of progesterone in culture medium were measured directly by RIA (17). Serum levels of LH were measured using an in-house immunofluorometric assay (Delfia, Wallac, Inc., Turku, Finland) as previously described (18). Levels of cAMP in culture medium were measured by RIA (19).3su%:), http://www.100md.com
Stereology and histology3su%:), http://www.100md.com
For stereology, testes were fixed in Bouin’s solution and stored in 70% ethanol. Tissue was embedded in Technovit 7100 resin (TAAB Laboratories Ltd., Reading, UK), cut into sections (20-µm thickness), and stained with Harris’ hematoxylin. The total testis volume was estimated using the Cavalieri principle (20), and the slides used to estimate the number of cells were also used to estimate testis volume. The optical disector technique (21) was used to count the number of Leydig cells in each testis. The numerical density of Leydig cells was estimated using an Olympus BX50 microscope fitted with a motorized stage (Prior Scientific Instruments, Cambridge, UK) and Stereologer software (Systems Planning Analysis, Alexandria, VA).
To generate semithin (1-µm) sections, testes were fixed in 1% glutaraldehyde-4% paraformaldehyde in phosphate buffer (0.1 M), pH 7.2, for 24 h at 4 C and embedded in araldite. Cut sections were stained with toluidine blue.n)#q2de, http://www.100md.com
Measurement of mRNA levelsn)#q2de, http://www.100md.com
Real-time PCR was used to measure levels of specific mRNA species present in the testes of control, FSHRKO, and FSHßKO animals (22). To allow results to be compared directly on a per testis basis, a standard amount of external control mRNA (luciferase) was added to each sample during the RNA extraction phase (22, 23). Sequences of primers and probes and the reaction conditions used were previously described (23).n)#q2de, http://www.100md.com
Cell transfectionsn)#q2de, http://www.100md.com
A mouse Leydig tumor cell line, mLTC-1 (24), was cultured in HEPES-buffered Waymouth’s medium (Sigma, St. Louis, MO) supplemented with 9% heat-inactivated horse serum (Life Technologies, Inc., Paisley, UK), 4.5% heat-inactivated fetal calf serum (BioClear, Calne, UK), and 50 mg/liter gentamicin (Biological Industries, Hemeek, Israel) at 37 C in 5% CO2. Cells were transiently transfected with an expression vector containing the mouse FSHR (pSG5-mFSHR-WT), the mouse FSHR with an inactivating point mutation (pSG5-mFSHR-C566T) (25, 26), or vector alone (pSG5) using Lipofectamine reagent (Life Technologies, Inc., Paisley, UK). Transfection efficiencies were verified by cotransfections with a luciferase expression vector. For transfections, cells were cultured on 10-cm diameter tissue culture plates to confluence of 50–70%, and 9 µg pSG construct vector or 0.3–0.6 µg luciferase vector were used to transfect the cells in Opti-MEM I with Glutamax (Invitrogen, Paisley, UK). After 5 h an equal volume of Waymouth’s medium supplemented with twice the normal concentration of serum was added to the cells. One day after the transfection cells were trypsinized and counted, and 60,000 cells/well were plated on 24-well plates. After 24 h cells were washed, and 1 ml Opti-MEM I with Glutamax plus 100 nmol/liter 1-methyl-3-isobutylxanthine (Alcon Laboratories, Inc., Milwaukee, WI) were added per well. Cells were incubated at 37 C in quadruplicate under basal conditions or in the presence of recombinant human FSH (National Hormone and Pituitary Program, McKesson BioServices, Rockville, MD). Aliquots of the incubation medium were collected after 2 h for cAMP and after 6 h for progesterone measurements.
Statistical analysis;, http://www.100md.com
Differences between control, FSHRKO, and FSHßKO animals were initially analyzed by single or two-factor ANOVA. Where a significant overall difference between animal groups was detected, differences between individual means were assessed by the Newman-Keuls test (after single factor analysis) or by t tests using the pooled variance (after two-factor analysis).;, http://www.100md.com
Results;, http://www.100md.com
Intratesticular testosterone levels;, http://www.100md.com
In control animals from both FSHßKO and FSHRKO groups testosterone levels per testis were low from birth to d 20, then increased markedly to d 60 and continued to rise up to d 180 (Fig. 1). In both FSHßKO and FSHRKO animals testosterone levels were normal in the prepubertal period up to d 20. After d 20 testosterone levels increased normally in the FSHßKO group, and on d 180 there was no significant difference from control values. In contrast, testosterone levels in FSHRKO mice increased more slowly after d 20 and were significantly less than control values on d 180 (Fig. 1).
fig.ommitteed/8dg5z, 百拇医药
Figure 1. Intratesticular testosterone concentrations in control heterozygous animals (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) animals during postnatal development. The results show testosterone levels per whole testis and represent the mean ± SEM of 4–17 animals/group. *, Significant (P < 0.05) difference from age-matched controls./8dg5z, 百拇医药
To measure the responsiveness of testes from FSHßKO and FSHRKO groups to exogenous stimulation by hCG, adult animals were injected with hCG as described in Materials and Methods. After hCG treatment, there was an increase in intratesticular testosterone levels in all groups, but levels in both FSHßKO and FSHRKO animals were significantly reduced compared with control values (Fig. 2). No difference was found between the two knockout models in their response to hCG./8dg5z, 百拇医药
fig.ommitteed/8dg5z, 百拇医药
Figure 2. Intratesticular testosterone levels per testis in adult control heterozygous mice (+/-) and in adult FSHßKO (-/-) and FSHRKO (-/-) mice after hCG injection. Animals were injected with hCG (0.5 IU/g) and were killed 1 h later. The mean ± SEM of four to nine animals per group are shown. Both 60- and 180-d-old animals were included in each group, but there was no age-dependent difference in testicular testosterone content after hCG injection, and data from different aged animals were pooled. Groups with different letter superscripts are significantly (P < 0.05) different.
Circulating LH levels47v, http://www.100md.com
Levels of circulating LH in adult FSHßKO mice did not differ significantly from those in control animals (Fig. 3). In contrast, LH levels in FSHRKO mice were significantly increased compared with control values (Fig. 3).47v, http://www.100md.com
fig.ommitteed47v, http://www.100md.com
Figure 3. Circulating LH levels in adult control heterozygous mice (+/-) and in adult FSHßKO (-/-) and FSHRKO (-/-) mice. The mean ± SEM of 4–12 animals/group are shown. The animals used in this study were 60 d old. Groups with different letter superscripts are significantly (P < 0.05) different.47v, http://www.100md.com
Expression of Leydig cell mRNA levels47v, http://www.100md.com
Levels of mRNA encoding the steroidogenic enzymes cytochrome P450 side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase type VI (3ßHSD VI), cytochrome P450 17{alpha} -hydroxylase (P450c17), and 17ßHSD III and the steriodogenic acute regulatory protein (StAR) were measured by real-time PCR. Levels of mRNA were measured relative to an externally added standard (luciferase) that allows direct comparison of mRNA levels between groups (22). For each mRNA species, mean levels in FSHRKO mice were lower than those in control animals, and this difference was significant for P450scc, 3ßHSD VI, and StAR (Fig. 4). In FSHßKO mice expression levels of the mRNA species measured were either normal (P450scc, 3ßHSD VI, and P450c17) or slightly, but significantly, increased (17ßHSD III and StAR).
fig.ommitteed@1h, http://www.100md.com
Figure 4. Measurement of Leydig cell-specific mRNA species by real-time PCR. The results show expression in control heterozygous mice (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) mice. RNA was extracted from whole testes of adult animals and reverse transcribed to generate cDNA. Levels of specific mRNA species were measured by real-time PCR and expressed relative to an external control (luciferase). As the same amount of luciferase mRNA was added to each sample, the results can be compared directly. Results show the mean ± SEM of four animals in each group. The animals used in this study were 180 d old. Groups with different letter superscripts are significantly (P < 0.05) different.@1h, http://www.100md.com
Testis size, Leydig cell number, and interstitial morphology@1h, http://www.100md.com
Testis size was reduced to about one third of normal in both FSHßKO and FSHRKO mice with the effect evident after d 5 (Fig. 5). Leydig cell number in both FSHßKO and FSHRKO mice was normal at birth and up to d 5 postnatally (Fig. 6). After d 5 Leydig cell number increased markedly in control animals, reaching a peak in adult animals. In FSHßKO mice Leydig cell development was normal after d 5, and numbers in the adult animal were not significantly different from control values. In contrast, Leydig cell numbers in FSHRKO mice failed to increase normally after d 5 and were significantly less than control values in the adult animal (Fig. 6). Despite differences in Leydig cell number interstitial cell morphology was similar in control, FSHßKO, and FSHRKO mice at the light microscopic level (Fig. 7.
fig.ommitteed*&f, 百拇医药
Figure 5. Changes in testicular volume during postnatal development in control heterozygous (+/-) mice and in FSHßKO (-/-) and FSHRKO (-/-) mice. Results show the mean ± SEM of three to five animals in each group.*&f, 百拇医药
fig.ommitteed*&f, 百拇医药
Figure 6. Leydig cell number during development in control heterozygous mice (+/-) and in FSHßKO (-/-) and FSHRKO (-/-) mice. Leydig cell number was measured using the optical dissector method. Results show the mean ± SEM of three to five animals in each group. *, Significant (P < 0.05) difference from age-matched controls.*&f, 百拇医药
fig.ommitteed*&f, 百拇医药
Figure 7. Light micrographs showing interstitial tissue in control, FSHßKO, and FSHRKO mice. The animals used to prepare these micrographs were 60 d old. Bar, 10 µm. The control animal used in this montage was an FSHß heterozygote. There were no clear differences in the morphology of the interstitial tissue between the different groups of animals at the light microscopic level.
Constitutive FSHR activity1w1f7tq, 百拇医药
To determine whether the FSHR shows constitutive activity, a Leydig cell line was transfected with either wild-type FSHR (mFSH-WT) or control vector, and the activity of the cells was measured. Monitoring by luciferase coexpression showed that transfection efficiencies with the different plasmids were similar in the individual experiments. The results in Fig. 8A show data from a single experiment in which cAMP levels were measured after transfection, and it is clear that there was a significant increase in basal cAMP production in the presence of the receptor and the absence of ligand. In contrast, transfection with an FSHR containing an inactivating point mutation (mFSH-C566T) had no effect on basal expression. As expected, transfection with mFSHR also conferred sensitivity to FSH, which was significantly reduced in cells transfected with mFSH-C566T. Figure 8B shows the combined results from five experiments designed to measure changes in basal cAMP and progesterone production by Leydig cells after transfection with the FSHR. Overall, there was an approximately 4-fold increase in both measures of basal cell activity after transfection with the receptor.
fig.ommitteed!w)4m+, 百拇医药
Figure 8. Progesterone and cAMP production in a Leydig cell line transfected with mFSHR (mFSHR-WT), mFSHR with an inactivating point mutation (mFSHR-C566T), or vector only (control). A, Results from a single experiment showing cAMP production by transfected cells under basal conditions and in the presence of increasing levels of hFSH. Results show the mean ± SEM of quadruplicate wells. B, Combined data from five experiments showing basal cAMP and progesterone production in cells transfected with mFSHR-WT or vector alone (control). Results show the mean ± SEM.!w)4m+, 百拇医药
Discussion!w)4m+, 百拇医药
FSH is a member of the heterodimeric glycoprotein hormone family, which includes LH, hCG, and TSH. Members of this family share a common {alpha} -subunit with biological specificity conferred by a hormone-specific ß-subunit, although only the heterodimers show biological activity. As with other members of the family, FSH acts through a single receptor type although considerable variation in the receptor can be induced through alternate splicing (26, 27). It might be expected, therefore, that the effects of induced null mutations in the hormone ß-subunit gene or the hormone receptor gene would induce similar phenotypes in the affected animals. Consistent with this, it has been shown that both FSHßKO and FSHRKO male mice are fertile, but have reduced testis size and reduced levels of spermatogenesis (13, 14, 15). The results reported here, however, show that there are significant differences in the effects of the mutations on Leydig cell development in affected animals.
Leydig cell development appears to be normal in FSHßKO mice, with no difference in Leydig cell number during development or in the adult animal and no difference in testosterone or circulating LH levels. This confirms previous reports showing that Leydig cell number and circulating testosterone levels are normal in adult FSHßKO mice (28). In contrast, testosterone production by FSHRKO mice was reduced despite an increase in circulating LH, and this was associated with reduced testicular expression of key mRNA species associated with steroidogenesis in Leydig cells. It is likely, however, that these apparent changes in Leydig cell function are due to a failure of normal Leydig cell proliferation/differentiation during puberty in FSHRKO mice, causing the numbers of cells in the adult animal to be reduced to about 60% of normal. This would explain the reduction in levels of intratesticular testosterone and Leydig cell-specific mRNA species and the rise in circulating LH, which is inversely linked to overall changes in testosterone production. In a recent study of another FSHRKO mouse model, testosterone levels were also significantly reduced in the adult animals (29), and it is likely that this is due to the same mechanism of action.
When a more marked phenotype arises from a receptor null mutation compared with a hormone null mutation, it is likely that this is either because the receptor is not specific to the hormone targeted with the mutation or because the receptor itself shows constitutive activity in the absence of agonist stimulation. In the case of the FSHR, there is as yet no evidence that a growth factor or hormone, other than FSH, can activate the receptor. Previous studies have shown, however, that G protein-coupled receptors can display constitutive activity (30), and studies reported here show clearly that the FSHR will express constitutive activity in the absence of ligand. The FSHR normally shows down-regulation in the presence of FSH (31), and it would be expected, therefore, that receptor levels will be increased in FSHßKO mice. As constitutive activity varies directly with receptor number, this will serve to increase activation of the cells and provides a clear explanation for phenotypic differences between FSHRKO and FSHßKO mice. In this respect the FSH and TSH receptors appear to show similar ligand-independent activation, which differs from the LH receptor, which is reported only to show ligand-independent activity under nonphysiological conditions (32).
Two generations of Leydig cells arise during normal testis development: a fetal population, which appears shortly after testis differentiation in utero, and an adult population, which arises shortly before puberty (starting around d 5–10 in the mouse) (33, 34). In both FSHßKO and FSHRKO mice, Leydig cell number and testicular testosterone levels were normal on d 1 and 5, showing that FSH and the FSHR do not appear to play a role in the development of the fetal population. During fetal growth in utero, Leydig cell function develops normally in GnRH-deficient mice (1) and in mice with a null mutation in the common {alpha} -subunit (35), and it is not unexpected, therefore, that Leydig cell function is normal on the day of birth in FSHßKO and FSHRKO mice. Shortly after birth, fetal Leydig cells become critically dependent on gonadotropins (1), but results reported here show that neither FSH nor its receptor is required at this time.3ak.9, http://www.100md.com
Within the testis only the Sertoli cell population expresses the FSHR, and the effect of the FSHR null mutation on Leydig cell development must, therefore, be mediated through Sertoli cells. This implies that factors released by Sertoli cells act to induce normal Leydig cell proliferation/differentiation. From other studies of mutant mice two Sertoli cell-derived factors (desert hedgehog and platelet-derived growth factor A) have been implicated in this process (36, 37). It is likely that the FSHR is a permissive factor in this process, ensuring that overall Sertoli cell activity is high enough to maintain normal output of the trophic factors. It is clear, however, that FSH is required for normal levels of Sertoli cell activity, because in both FSHßKO and FSHRKO mice cell number is reduced, and spermatogenesis is compromised (28, 38).
Given that LH levels, Leydig cell number, and testosterone levels are normal in FSHßKO mice, it is not clear why intratesticular testosterone levels fail to increase normally after hCG stimulation. It may simply be due to a physical effect of the 50% reduction in size of the FSHßKO testis, which will affect blood flow to the testis interstitium and may limit the amount of testosterone that can actually be held within the testis. Alternatively, it is possible that the sensitivity of the testis to hCG stimulation or the maximum steroidogenic capacity of the Leydig cells is reduced in the absence of FSH. This does not, however, appear to affect androgen production under normal conditions.zp?c, http://www.100md.com
Previous studies showing an effect of FSH injections on Leydig cell activity were carried out using animals with low or absent circulating LH (4, 5, 6, 7, 8, 9, 10, 11). Results from this study suggest that in the presence of LH, FSH is not required for normal Leydig cell development as long as FSHR are present. The lack of effect of the FSHß null mutation on Leydig cell number contrasts with the almost complete failure of postpubertal development of Leydig cell number in GnRH-deficient mice, which lack LH and FSH (39). Thus, LH alone is sufficient for normal postnatal Leydig cell development (in the presence of the FSHR), but in the absence of LH, evidence from earlier studies suggests that FSH can induce stimulation of Leydig cell activity (9, 11).
Received June 20, 2002.q\n, 百拇医药
Accepted for publication September 13, 2002.q\n, 百拇医药
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