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Endocrinology, doi:10.1210/en.2007-0837
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Endocrinology Vol. 148, No. 12 6019-6025
Copyright © 2007 by The Endocrine Society

Age-Related Alterations in Pituitary and Testicular Functions in Long-Lived Growth Hormone Receptor Gene-Disrupted Mice

Varadaraj Chandrashekar1, Christina R. Dawson, Eric R. Martin, Juliana S. Rocha, Andrzej Bartke and John J. Kopchick

Department of Physiology (V.C., C.R.D., E.R.M., A.B.), Southern Illinois University School of Medicine, Carbondale, Illinois 62901; Internal Medicine (J.S.R., A.B.), Southern Illinois University School of Medicine, Springfield, Illinois 62794; and Biomedical Sciences Department and Edison Biotechnology Institute (J.J.K.), College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701

Address all correspondence and requests for reprints to: Andrzej Bartke, Ph.D., Department of Internal Medicine, Geriatrics Research, Southern Illinois University School of Medicine, Springfield, Illinois 62794. E-mail: abartke{at}siumed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The somatotropic axis, GH, and IGF-I interact with the hypothalamic-pituitary-gonadal axis in health and disease. GH-resistant GH receptor-disrupted knockout (GHRKO) male mice are fertile but exhibit delayed puberty and decreases in plasma FSH levels, testicular content of LH, and prolactin (PRL) receptors, whereas PRL levels are elevated. Because the lifespan of GHRKO mice is much greater than the lifespan of their normal siblings, it was of interest to compare age-related changes in the hypothalamic-pituitary-gonadal axis in GHRKO and normal animals. Plasma IGF-I, insulin, PRL, LH, FSH, androstenedione and testosterone levels, and acute responses to GnRH and LH were measured in young (2–4 and 5–6 months of age) and old (18–19 and 23–26 months of age) male GHRKO mice and their normal siblings. Plasma IGF-I was not detectable in GHRKO mice. Plasma PRL levels increased with age in normal mice but declined in GHRKO males, and did not differ in old GHRKO and normal animals. Plasma LH responses to acute GnRH stimulation were attenuated in GHRKO mice but increased with age only in normal mice. Plasma FSH levels were decreased in GHRKO mice regardless of age. Plasma testosterone responses to LH stimulation were attenuated in old mice regardless of genotype, whereas plasma androstenedione responses were reduced with age only in GHRKO mice. Testicular IGF-I mRNA levels were normal in young and increased in old GHRKO mice, whereas testicular concentrations and total IGF-I levels were decreased in these animals. These findings indicate that GH resistance due to targeted disruption of the GH receptor gene in mice leads to suppression of testicular IGF-I levels, and modifies the effects of aging on plasma PRL levels and responses of the pituitary and testes to GnRH and LH stimulation. Plasma testosterone levels declined during aging in normal but not in GHRKO mice, and the age-related increase in the LH responses to exogenous GnRH was absent in GHRKO mice, perhaps reflecting a delay of aging in these remarkably long-lived animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS CONSIDERABLE evidence for complex, reciprocal interactions between the somatotropic axis, GH, and IGF-I, and the hypothalamic-pituitary-gonadal (HPG) axis in both sexes (1, 2). After our earlier studies of the effects of GH treatment in hypopituitary mutant mice and GH overexpression in transgenic mice on testicular function (3, 4, 5), we have examined the effects of GH resistance on the HPG axis in male GH receptor-disrupted knockout (GHRKO) mice. These studies provided evidence for delayed puberty (6) and numerous hormonal alterations in young adults, including reduced FSH levels, increased prolactin (PRL) levels, reduced testicular content of LH and PRL receptors, and attenuated responses of the pituitary and testes to stimulation with exogenous GnRH and LH, respectively (7, 8). Because GHRKO mice are characterized by a remarkable increase in longevity (9) and various indices of delayed aging (10, 11), it was of interest to determine whether the effects of aging on the HPG axis are altered in these animals, and examine pituitary and testicular function in old GHRKO males.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
GHRKO mice (–/–) were produced as described previously (7). Adult normal female mice (+/–) were mated with either GHRKO (–/–) or +/– male mice, and the resulting GHRKO mice and non-GHRKO siblings (normal mice) were used in the present study. These animals are on a heterozygous genetic background derived from the Ola-BALB/cJ, C57BL/6J, and C3H/J strains. We previously found no differences in body weight (BW) and plasma IGF-I levels between homozygous (+/+) and heterozygous (+/–) normal mice (7), and it was reported that these two genotypes are not significantly different in terms of secretion of GH and GH binding protein, and other endocrine parameters (13). In the present study, mice were classified as GHRKO or normal based on BW, and this was verified by results of RIA measurement of plasma IGF-I levels. All mice were housed in a room with a controlled photoperiod of 12-h light per day (lights on from 0600–1800 h) and a temperature of 22–23 C. Mice were given free access to a nutritionally balanced diet (LabDiet; Purina Mills, St. Louis, MO) and tap water. In the present study, mice at and over 18 months of age will be referred as "old" or "aged" mice. The experimental protocols were approved by the Animal Care Committee of Southern Illinois University.

Experiment 1: gonadotropin and PRL levels, and LH responses to GnRH stimulation
Young (2–3 and 5–6 months of age) and aged (18–19 and 23–26 months of age) male GHRKO mice and their normal siblings were injected ip with saline or GnRH in saline (1 ng/g BW, lot no. 106T-58302; Sigma Chemical Co., St. Louis, MO) using six to 10 mice per group. We have shown previously that this dose of GnRH is effective in inducing LH secretion in normal mice (7, 14). Fifteen minutes after saline or GnRH injection, the animals were anesthetized with isoflurane (Baxter Healthcare Corp., Deerfield, IL), and blood was obtained via heart puncture. Plasma samples were frozen at –20 C until assayed for IGF-I, insulin, and LH levels. The testes and seminal vesicles (with secretions) were removed and weighed.

Another batch of young (2–6 months of age) and old (26–29 months of age) GHRKO mice and their normal siblings (seven to 12 mice per subgroup) was used to assess the basal FSH and PRL levels. Testes were used to assess IGF-I mRNA and IGF-I levels.

Experiment 2: effects of LH on testicular function
Young (3–4 months of age) and old (18–19 months of age) GHRKO mice and their normal siblings were treated (single ip injection) with either saline or ovine LH (oLH) (National Institutes of Health oLH26; 0.3 µg/g BW) dissolved in saline or with saline alone, using eight to 10 mice per group. This dose of LH was effective in increasing the circulating androgen levels in mice in our previous studies (13, 14). One hour after saline or LH administration, the animals were anesthetized with isoflurane, and blood was obtained via heart puncture. Plasma samples were stored at –20 C until assayed for androstenedione and testosterone levels by RIAs, as described previously (14, 15). We have previously reported differential effects of GH receptor (GHR) disruption on androstenedione and testosterone responses to LH in young adult GHRKO mice (8), indicating that enzymatic conversion of androstenedione to testosterone may be altered in the testes of these animals.

Extraction of mRNA and cDNA synthesis
Total RNA was extracted from the testes of young (2 months of age) and old (26–29 months of age) GHRKO and normal mice (n = 7–12 mice per group) by the guanidinium thiocyanate-phenol-chloroform method (16). One microgram of total RNA was electrophoresed on a 1.5% agarose gel to confirm RNA integrity. cDNA was made using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) following the manufacturer’s protocol.

Real-time PCR
The real-time PCR procedure was performed as previously described (17). In brief, the real-time PCR amplification was performed with the iQ SYBR Green PCR Super Mix (Bio-Rad Laboratories) using the SmartCycler (Cepheid, Sunnyvale, CA). The IGF-I primers used were 5'-CTGAGCTGGTGGATGCTCTT (forward) and 5'-CACTCATCCACAATGCCTGT (reverse), as designed by Masternak et al. (18). The RT-PCR program included a 95 C denaturation step for 2 min, followed by 45 cycles of 95 C denaturation for 15 sec, 62 C annealing for 30 sec, and 72 C extension for 30 sec. Detection of fluorescent product was performed at the end of the 72 C extension period. Melting curve and agarose gel electrophoresis were used to confirm the identity of PCR products. The expression of ß-2-microglobulin was used as a housekeeping gene (18). Relative expression from real-time PCR data was quantified using Cepheid SmartCycler software, as previously described (17).

Hormone assays
Plasma and testicular IGF-I concentrations were measured by RIA as described by us and others (19, 20, 21). The presence of IGF-I binding proteins interferes in the RIA procedure. Therefore, these proteins were removed from the plasma and the testicular homogenates. Plasma samples and testicular homogenates were extracted with formic acid and acetone as described previously (20, 22). This extraction method does not completely eliminate all IGF-I binding proteins (21). Therefore, to remove the remaining IGF-I binding proteins, acid-acetone extracts were subjected to cryoprecipitation, a procedure described previously. The mean recoveries of iodinated IGF-I added to the plasma were 94.2%. The Tris-neutralized extracts were diluted with RIA buffer containing 0.02% protamine sulfate and 0.05% Tween 20. Diluted plasma extracts were used in this RIA. The purified recombinant human IGF-I preparation purchased from Amgen Biologicals (Thousand Oaks, CA) was used as the reference preparation, and the human IGF-I (no. A52-8MH-144; Eli Lilly and Co., Indianapolis, IN) was iodinated and used as trace. Antiserum prepared against human IGF-I (no. UB2-495; developed by Drs. L. E. Underwood and J. J. Van Wyk, University of North Carolina at Chapel Hill, NC) was used in this RIA. Varying quantities of the mouse plasma extract pool produced a curve parallel to the curve obtained by varying amounts of human IGF-I preparation, thus validating the use of these human IGF-I RIA reagents to measure IGF-I levels in the mouse. The sensitivity of this assay was 32 pg/tube. All plasma or testicular samples were included in the same assay to avoid interassay variability. The intraassay coefficient of variation was 3.8%.

Circulating insulin levels were assayed using sensitive rat insulin RIA reagents purchased from LINCO Research, Inc. (St. Charles, MO). The insulin antibody used in this assay cross-reacted 100% with mouse insulin. The sensitivity of this assay was 10 pg/tube. All plasma samples were included in the same assay, and the intraassay coefficient of variation was 6.2%.

Plasma PRL concentrations were measured by RIA as previously described (23). Briefly, mouse PRL reference preparation (AFP-6476C) and mouse PRL antiserum (AFP-131078; both kindly provided by Dr. A. F. Parlow, Pituitary Hormone and Antisera Center, Harbor-University of California Los Angeles Medical Center, Torrance, CA) were used in this PRL assay. All plasma samples were measured starting on the same day, using the same-day diluted reference preparation, antiserum, and repurified hormone trace. The sensitivity of this assay was 0.1 ng/tube, and the intraassay coefficient of variation was 4.1%.

Plasma LH and FSH levels were determined by RIAs as described previously (14, 23) using reagents kindly supplied by Drs. A. F. Parlow and G. D. Niswender (Colorado State University, Fort Collins, CO), and National Hormone and Pituitary Program, Rockville, MD, National Institutes of Health, Bethesda, MD. Briefly, recombinant LH RP-2 reference preparation, oLH antiserum (GDN-15), recombinant FSH (rFSH) RP-2 reference preparation, and rFSH antiserum (S-11) were used in LH and FSH RIAs, respectively. Various amounts of pooled plasma obtained from intact and castrated mice produced curves parallel to those of varied amounts of recombinant LH and rFSH reference preparations, validating the use of these reagents for measurements of LH and FSH levels in mice. The sensitivities of these assays were: LH, 0.010 ng/tube; and FSH, 0.250 ng/tube. All plasma samples of each experiment were measured starting on the same day, using the same-day diluted reference preparation, antiserum and repurified hormone trace. The intraassay coefficients of variation were 2.0% for LH and 2.8% for FSH.

Plasma androstenedione and testosterone levels were determined by RIAs as described previously (14, 15), with a standard extraction procedure using anhydrous diethyl ether. The androstenedione antibody (X-322) used to measure plasma androstenedione levels was purchased from the Southwest Foundation for Biomedical Research (St. Antonio, TX), and it cross-reacted 2.5% with testosterone. The testosterone antiserum (GDN-S250) used in testosterone RIA was kindly donated by Dr. G. D. Niswender, and it cross-reacted 1.5% with androstenedione. The sensitivities of these assays were 10 pg/tube for androstenedione and 5 pg/tube for testosterone. For a specific assay, all samples were assayed on the same day using the same-day diluted specific antiserum and the radiolabeled steroid. The mean intraassay coefficients of variation were 6.6% for androstenedione and 4.8% for testosterone.

Statistical analyses
Statistical analysis was performed by ANOVA, followed by Student-Neuman-Keuls test. The Student’s t test was used when the values of two groups were compared. A P value less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body, testicular, and seminal vesicle weights
In all age groups, the average, absolute body, testicular, and seminal vesicle weights were significantly (P < 0.001) lower in GHRKO mice relative to their normal siblings (Table 1Go).


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  		TABLE 1. Effects of aging on BW, testicular, and seminal vesicle (SV) weights in normal and GHRKO mice

 
Plasma IGF-I and insulin levels
The circulating IGF-I levels were not detectable in young and old GHRKO mice, whereas the plasma levels of this hormone in aged and young normal siblings were not significantly different (Fig. 1Go). Circulating insulin levels were significantly (P < 0.001) decreased in both young and old GHRKO mice relative to young and old normal siblings (Fig. 2Go).


Figure 1
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  		FIG. 1. Circulating IGF-I levels in young (5–6 months of age) and old (23–26 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05. ND, Not detectable.

 

Figure 2
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  		FIG. 2. Circulating insulin levels in young (5–6 months of age) and old (23–26 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
Plasma PRL levels
Plasma PRL levels were significantly (P < 0.001) increased in young GHRKO mice compared with young normal siblings. There were significant (P < 0.001) increases in plasma PRL concentrations in aged normal compared with young normal mice. In contrast, plasma PRL levels were decreased (P < 0.025) in old GHR gene-disrupted mice relative to young GHRKO mice. These changes resulted in nearly identical plasma PRL levels in old normal and old GHRKO mice (Fig. 3Go).


Figure 3
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  		FIG. 3. Circulating PRL levels in young (3–6 months of age) and old (26–29 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
Experiment 1: effect of GnRH on plasma LH and basal FSH levels in young and old mice
The basal circulating LH levels were similar in young and old normal and GHRKO mice. Treatment with a single injection of GnRH resulted in a significant (P < 0.001) increase in plasma LH levels in both types of mice, regardless of age. However, these LH responses were significantly (P < 0.001) attenuated in young and old GHRKO mice relative to normal siblings (Fig. 4Go). In addition, the plasma LH levels were significantly higher (P < 0.01) in old normal mice treated with GnRH than in similarly treated young normal mice. In contrast, this plasma LH response to GnRH treatment was not different in young vs. old GHRKO mice.


Figure 4
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  		FIG. 4. Circulating LH levels in young and aged GHRKO mice and their normal siblings injected with either saline or GnRH in saline. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
The basal plasma FSH levels in both young and old GHRKO mice were significantly (P < 0.001) lower than in their normal siblings (Fig. 5Go).


Figure 5
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  		FIG. 5. Circulating FSH levels in young (3–6 months of age) and old (26–29 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
Experiment 2: plasma androstenedione and testosterone responses to LH treatment
Basal plasma androstenedione levels were similar in young and old normal, as well as GHRKO mice. Treatment with LH significantly (P < 0.001) increased plasma androstenedione levels in both normal and GHRKO mice, regardless of their age. However, androstenedione levels in LH-injected old GHRKO mice were significantly lower (P < 0.01) than in young mice of either genotype or in old normal mice given identical treatment (Fig. 6Go).


Figure 6
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  		FIG. 6. Circulating androstenedione (A-DIONE) levels in young (3–4 months of age) and old GHRKO (18–19 months of age) mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
Basal testosterone concentrations were decreased (P < 0.005) in aged normal compared with young normal mice, whereas no age-related changes in plasma testosterone levels were detected in GHRKO mice. As expected, LH treatment significantly (P < 0.001) increased plasma testosterone levels in both young and aged, normal and GHRKO mice. Relative to young normal siblings, this testosterone response was attenuated in aged normal (P < 0.01), as well as in young (P < 0.001) and aged GHRKO (P < 0.001) mice. In addition, the already decreased plasma testosterone response to LH treatment in young GHR gene-disrupted mice was further reduced (P < 0.001) in aged GHRKO mice (Fig. 7Go).


Figure 7
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  		FIG. 7. Circulating testosterone (T) levels in young (3–4 months of age) and old (18–19 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 
Testicular IGF-I mRNA and IGF-I levels
IGF-I mRNA was detected in the testes of both young and old, normal and GHRKO mice. However, testicular IGF-I expression was higher in the testes of old GHRKO mice relative to young GHRKO mice (P < 0.025), and young and old normal mice (P < 0.05; Fig. 8Go). Although IGF-I was undetectable in the plasma samples obtained from GHRKO mice, low IGF-I levels were measurable in the testes of these animals, regardless of age. Testicular IGF-I levels were significantly (P < 0.001) lower in GHRKO mice relative to normal siblings (Figs. 9Go and 10Go).


Figure 8
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  		FIG. 8. Relative IGF-I mRNA expression in the testes of young (2 months of age) and old (26–29 months of age) GHRKO and their normal siblings. The RT-PCR data were normalized to housekeeping gene ß-2-microglobulin and expressed as the relative expression. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 

Figure 9
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  		FIG. 9. Testicular IGF-I concentrations (pg/mg testis) in young (3–6 months of age) and old (26–29 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 

Figure 10
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  		FIG. 10. Total testicular IGF-I levels (ng/two testes) in young (3–6 months of age) and old (26–29 months of age) GHRKO mice and their normal siblings. Means ± SEM. Values without the same letter differ at a significance level of at least P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The key finding of the present study is that some of the age-related alterations in the function of the HPG axis were absent in the long-lived, GH-resistant mice. In contrast to their normal siblings, the GHRKO mice did not experience the decline in plasma testosterone levels or the increase in pituitary LH responses to GnRH stimulation. Moreover, GHRKO mice experienced an age-related decrease in plasma PRL levels, a change opposite to the effects of aging in normal animals, and reacted to LH stimulation by attaining lower androstenedione levels than those measured in old normal LH-injected mice. The lack of an age-related increase in LH response to GnRH stimulation in GHRKO mice likely reflects the persistence of unaltered negative feedback by testicular testosterone during aging in these animals. Failure to detect age-related changes in several parameters of endocrine function in the GHRKO mice in the present study might reflect either an absence or temporal delay of these changes.

GH and IGF-I have a well-documented role in both somatic growth and reproductive development. As expected, the average BW and the weights of the testes and seminal vesicles were significantly reduced in GHRKO compared with normal mice. These differences were evident in all age groups. It has been shown previously that the growth of the testis is delayed in rats lacking GH secretion (24), and in rats treated with GHRH antiserum (25), suggesting that GH/IGF-I plays an important role in the growth of the testis. Growth of the seminal vesicles is androgen dependent. Because the basal circulating testosterone levels were similar in both normal and GHRKO mice, we speculate that either the number of androgen receptors is reduced in the seminal vesicle of these animals, or sensitivity to androgen action is reduced by other mechanisms, perhaps including profound suppression of circulating IGF-I levels.

Although circulating GH levels are increased in GHRKO mice, circulating IGF-I levels are dramatically reduced, reflecting the absence of GHRs (Refs. 7 , 8 , and 13 and the present data). Whereas the basal circulating LH levels were not affected in GHR gene-disrupted mice, the GnRH effect on the plasma LH levels was significantly decreased in both young and aged GHRKO mice, suggesting that systemic IGF-I is required for the normal gonadotropin response to GnRH stimulation. However, the reduced plasma LH response in GHRKO mice was not further modified by age, whereas in normal mice, LH levels measured after GnRH administration increased with age. We have previously shown that induction of endogenous IGF-I secretion by GH administration improves the GnRH action on LH secretion in hypopituitary Ames dwarf mice, in which plasma IGF-I levels are also greatly suppressed (4). Plasma LH responses to GnRH treatment were increased in aged normal siblings. It was previously demonstrated that the LH response to GnRH treatment was significantly greater in old compared with young female mice (26). However, it was also reported that the anterior pituitaries of the normal aged female mice and aged male rats were less sensitive to GnRH stimulation (27, 28). In aged men, circulating gonadotropin levels are increased (29, 30, 31), whereas the LH pulse amplitude (a reflection of the amount of LH secreted per burst) is decreased (32). Furthermore, pituitary sensitivity to GnRH treatment in terms of LH secretion is increased in older men. Similarly, our data indicate that the pituitaries of aged normal male mice are more sensitive to GnRH action than the pituitaries of young adult animals.

In both young and old GHRKO mice, plasma FSH levels were significantly lower than in their normal siblings, suggesting that secretion of inhibin by the testes may have been increased (33, 34). Aging did not further affect the already suppressed FSH secretion in GHRKO mice. Reduced FSH secretion likely contributes to the reduction of testicular weight in GHRKO mice.

As expected from our previous studies (7, 8), PRL levels were increased in young male GHRKO mice relative to young normal mice. It is interesting that a similar situation was observed in GH-resistant Laron syndrome patients and was thought to be due to a "drift" phenomenon of mammosomatotropes, which produce large amounts of GH (35). "Laron dwarf" (GHRKO) mice, similarly to Laron syndrome patients, secrete large amounts of GH (13).

Surprisingly, in GHRKO mice, plasma PRL levels were declining rather than increasing with age. Further studies will be needed to identify the possible mechanism of differential effects of aging on PRL levels in GHRKO compared with normal mice. In old normal mice, plasma PRL levels were increased, resembling the observations in aging men (36).

A number of in vitro studies have shown that IGF-I can influence testicular function. IGF-I has increased testosterone release from the isolated Leydig cells (37, 38, 39). In the present study, disruption of the GHR gene and the resulting suppression of peripheral IGF-I levels were associated with reduced plasma testosterone response to exogenous LH, confirming our previous observations (8). Aging resulted in decreased plasma testosterone responses to injected LH in both normal and GHRKO mice. In contrast, plasma androstenedione responses to LH were significantly reduced during aging in GHRKO but not in normal males. Presumably, the activity of 17ß-hydroxysteroid dehydrogenase is differentially affected by aging in GHRKO and normal mice. Data obtained in rats (40) encourage speculation that this may have been related to differential effects of aging on plasma PRL levels in these animals.

Because the present study showed no significant age-related differences in the circulating IGF-I levels in either normal or GHRKO mice, we have examined IGF-I expression in the testes of these animals. In young adult GHRKO mice, the levels of testicular IGF-I mRNA did not differ from the levels measured in young normal mice, whereas in old GHRKO males, testicular IGF-I mRNA levels were slightly, but significantly, higher than in normal old mice. In contrast, both concentration and content of IGF-I were reduced in GHRKO, as compared with normal mice, regardless of age. Discrepancies between the alterations in the steady-state levels of IGF-I mRNA and the levels of IGF-I protein in the GHRKO testes may relate to altered stabilization of the message, reduced translation, or enhanced degradation of IGF-I. Testicular IGF-I production is known to be influenced by gonadotropins (41, 42). Therefore, it can be suspected that the reduced testicular IGF-I in GHRKO mice is due to a decrease in FSH levels. In support of this possibility, FSH treatment increased testicular IGF-I levels (43).

Interestingly, aging was associated with an increase of testicular IGF-I levels in GHRKO but not in normal mice. These findings indicate that posttranscriptional processing of IGF-I mRNA is altered in GHRKO mice and that aging affects testicular IGF-I expression in these animals. Reduction in local (testicular) IGF-I levels in GHRKO compared with normal mice may have contributed to reduced testosterone responses to LH in these animals. However, the age-related decrease in testosterone responses to LH in both normal and GHRKO must have been due to other causes because testicular IGF-I levels were either maintained or increased with age. A reduction in the number of LH binding sites that was described in aging Brown-Norway rats (44) could provide an explanation for attenuated testosterone responses to LH. This, in turn, may reflect an age-related decline in the number of Leydig cells (12, 45).

In summary, aging exerts differential effects on pituitary responses to GnRH, plasma PRL and testosterone levels, testicular responses to LH, and local expression of IGF-I in the testis in GH-resistant GHRKO mice compared with normal animals. Maintenance of unaltered basal testosterone levels and absence of an increase in LH responses to GnRH in old GHRKO animals suggest that age-related alterations in the endocrine function of the HPG axis are absent, attenuated, or, more likely, significantly delayed in these remarkably long-lived animals.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD-37950 (to V.C.) and AG-19899 (to A.B.). J.J.K. was supported in part by the State of Ohio’s Eminent Scholar Program, which included a gift by Milton and Lawrence Goll and by DiAthegen, LLC. J.S.R. is affiliated with the Department of Morphology, Laboratory of Cellular Biology, Institute of Biological Sciences/Federal University of Minas Gerais, Belo Horizonte-MG 31270-901, Brazil, and was supported by scholarships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Fundação de Amparo à Pesquisa do Estado de Minas Gerais.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 13, 2007

1 This paper is dedicated to V.C.’s memory. Back

Abbreviations: BW, Body weight; GHR, GH receptor; GHRKO, GH receptor-disrupted knockout; HPG, hypothalamic-pituitary-gonadal; oLH, ovine LH; PRL, prolactin; rFSH, recombinant FSH.

Received June 22, 2007.

Accepted for publication September 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Veldhuis JD, Roemmich JN, Richmond EJ, Bowers CY 2006 Somatotropic and gonadotropic axes linkages in infancy, childhood, and the puberty-adult transition. Endocr Rev 27:101–140[Abstract/Free Full Text]
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