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Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-6512
Address all correspondence and requests for reprints to: Dr. V. Chandrashekar, Department of Physiology, Life Science II Building, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-6512.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of exogenous GH
Rats were divided into two groups and implanted sc with osmotic
pumps (one pump per rat; model 2ML2, lot 043103, Alza Corp., Palo Alto,
CA) filled with either alkaline saline (pH 12) or bovine GH (bGH; USDA,
B-1; 10.42 µg/h in alkaline saline; a total of 250 µg/day). It has
been shown that bGH has less immunogenic effect than other GH
preparations (11), and its antigenicity is similar to that of rat GH
(rGH) (12). For these reasons we opted to use bGH for these studies.
The following experiments were conducted. In Exp 1, on day 14, each
group of rats was divided into two subgroups (n = 9–11
rats/subgroup) and treated ip with either saline or GnRH in saline (1
ng/g BW; lot 016441, Peninsula Laboratories, Belmont, CA). Fifteen
minutes after saline or GnRH treatment, rats were anesthetized with
ether, and blood was obtained via heart puncture. Plasma samples were
frozen at -20 C. In Exp II, on day 7 after saline or bGH
administration via osmotic pumps, rats were bilaterally castrated under
ether anesthesia. On the same day, each group of rats was divided into
two subgroups (n = 10–12 rats/subgroup) and injected sc on
alternate days with either peanut oil or testosterone propionate (TP; 1
µg/g BW) in peanut oil. On days 1, 7, 8, 10, 12, and 14, blood was
collected as described in Exp I, and plasma samples were stored at -20
C. Plasma LH, PRL, FSH, bGH, bGH antibodies, and insulin-like growth
factor I (IGF-I) were measured by RIAs. In Exp I, plasma testosterone
levels were also measured by RIA.
Effects of active immunization against GH
Rats were actively immunized with oGH (100 µg/rat; NIDDK
oGH-15) in alkaline saline-complete Freund’s adjuvant mixture (1:1).
Control rats were injected with alkaline saline-complete Freund’s
adjuvant. Four weeks after this primary injection, rats were injected
(secondary injections) with either alkaline saline-incomplete Freund
adjuvant or oGH (same dose) in the saline-incomplete adjuvant once a
month for a duration of 5 (Exp III) or 6 (Exp IV) months. Injections
were made sc at multiple sites in the back of the rats. It has been
shown that rGH and oGH have similar antigenic structures (12).
Therefore, we opted to immunize rats against oGH. The following
experiments were conducted. In Exp III, 2 weeks after five secondary
injections of either saline-adjuvant (adjuvant-injected) or GH in
saline-adjuvant mixture (GH-immunized), rats were divided into two
subgroups (n = 8–12 rats/subgroup) and injected (single ip
injection) with either saline or GnRH (500 ng/rat; lot 106F-58302,
Sigma Chemical Co., St. Louis, MO) in saline. The mean body weight of
these animals was 531.3 ± 7.5 g. Therefore, this dose of
GnRH was similar to that used in Exp I. Fifteen minutes after saline or
GnRH injection, blood was obtained via heart puncture under ether
anesthesia. Plasma samples were stored at -20 C until assayed for
circulating GH antibody titers and hormone levels. In Exp IV, 2 weeks
after six secondary injections of either saline-adjuvant or GH in
saline-adjuvant mixture, rats were bilaterally castrated under ether
anesthesia. These castrated rats were divided into two subgroups
(n = 8–12 rats/subgroup) and injected sc on alternate days with
either peanut oil or TP (100 µg/100 g BW) in peanut oil. Blood was
obtained from these animals via heart puncture on days 1, 2, 4, 6, and
8 after castration as in Exp III. Plasma samples were stored at -20 C
until assayed for circulating GH antibody titers and hormone levels.
Circulating GH antibody titers and plasma LH, FSH, PRL, and IGF-I
levels were determined by RIAs. Plasma samples obtained from Exp III
were also used to measure bGH and testosterone levels by RIA.
Detection of circulating GH antibodies
Circulating GH antibody titers were determined as described
previously (13). Briefly, plasma samples, obtained from rats injected
with saline-adjuvant or from those actively immunized against GH were
incubated at a final dilution of 1:1000 with a mixture of EDTA,
phosphosaline buffer, and trace amounts (2–3 ng/tube) of
[125I]rGH at 4 C for 48 h, and the labeled
antigen-antibody complex was precipitated by the addition of antirat
-globulin (Calbiochem Corp., La Jolla, CA). After further incubation
for 20–24 h at 4 C, the precipitates were separated by centrifugation
at 1200 x g for 30 min, and the radioactivity was
measured in an automatic -spectrometer.
To assess the possibility of the development of antibodies against the bGH infused via osmotic pumps, plasma samples from bGH-treated and saline-infused rats were incubated at a final dilution of 1:125 with trace amounts of [125I]bGH as described above. As a positive binding control, [125I]bGH was incubated with a bGH antiserum (AFP-55, provided by Dr. A. F. Parlow) at a final dilution of 1:250,000.
Hormone assays
The plasma concentrations of LH, FSH, and PRL levels were
determined by specific homologous RIAs as described previously (14),
using reagents generously supplied by Dr. A. F. Parlow and
National Hormone and Pituitary Program, NIH. Briefly, rLH RP-2
reference preparation and rLH S-8 antiserum, rFSH RP-2 reference
preparation and rFSH S-11 antiserum, and rPRL RP-3 reference
preparation and rPRL S-9 antiserum were used in LH, FSH, and PRL RIAs,
respectively. The sensitivities of these assays were: LH, 0.025; FSH,
0.250; and PRL, 0.050 ng/tube. For each hormone assay, all plasma
samples from a particular experiment were measured starting on the same
day, using the same day diluted reference preparation, antiserum, and
repurified hormone trace. The mean intraassay coefficients of variation
were 3.0% for LH, 4.7% for FSH, and 7.2% for PRL.
Plasma testosterone levels were determined by RIA as described previously (7, 13) with a standard extraction (extracted with anhydrous diethyl ether) procedure. The sensitivity of this testosterone assay was 5 pg/tube. The mean intraassay coefficient of variation was 2.2%.
Plasma bGH levels in rats infused with either saline or bGH via osmotic pumps were determined by a homologous RIA using reagents provided by Dr. A. F. Parlow (Harbor-University of California-Los Angeles, Torrance, CA). Briefly, highly purified bGH (AFP-7698B) was used for iodination and as a reference preparation. bGH antiserum (AFP-55) was used in this RIA. The sensitivity of this assay was 50 pg/tube. All plasma samples were included in the same assay, and the intraassay coefficient of variation was 4.5%.
Plasma IGF-I concentrations were measured by RIA as described by us and others (7, 15, 16). As the presence of IGF-I-binding proteins in the plasma interferes in the RIA procedure, these proteins were removed from the plasma. Plasma samples were extracted with formic acid and acetone as described previously (15). Because this extraction method does not eliminate all IGF-I-binding proteins present in the plasma (16), acid-acetone extracts were subjected to cryoprecipitation, a procedure described previously (16). The mean recoveries of iodinated IGF-I added to the plasma were 85.8%. The Tris-neutralized plasma 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 (A52-8MH-144, Eli Lilly Co., Indianapolis, IN) was iodinated and used as the trace. Antiserum prepared against human IGF-I (UB2-495, developed by Drs. L. E. Underwood and J. J. Van Wyk, University of North Carolina-Chapel Hill) was used in this RIA. Varying quantities of the rat plasma extract pool produced a curve parallel to that obtained by varying amounts of human IGF-I preparation. Therefore, it is valid to use these human IGF-I RIA reagents to measure IGF-I levels in rat plasma. The sensitivity of this assay was 32 pg/tube. All plasma extracts were included in the same RIA to avoid interassay variability. The intraassay coefficient of variation was 1.8%.
Statistical analysis
Statistical analyses were performed by ANOVA followed by the
Student-Newman-Keuls test. Student’s t test was used when
two values were compared.
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Results |
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The circulating bGH antibodies were not detected in rats infused with bGH via osmotic pumps. The percent binding of [125I]bGH was 5.9 ± 0.1 for saline-infused rats and 5.7 ± 0.1 for bGH-treated rats. (There was a binding of 42.7% with bGH antibody AFP-55, generated by Dr. Parlow).
Plasma bGH levels in rats infused with bGH
Plasma bGH was detected in rats treated with bGH (75.6 ± 4.2
ng/ml), whereas bGH was not detectable in rats infused with saline.
Circulating IGF-I levels
Plasma IGF-I levels were significantly (P <
0.001) decreased after immunization with GH (adjuvant-injected rats,
310.00 ± 8.2 ng/ml; GH-immunized animals, 224.53 ± 4.4
ng/ml). In rats infused with saline, the plasma IGF-I levels were
394.01 ± 7.0 ng/ml and were modestly decreased (341.64 ±
12.5 ng/ml; P < 0.005) in rats infused with bGH for 14
days.
Exp I: effects of GnRH on plasma gonadotropin, PRL, and
testosterone levels in GH-treated gonad-intact rats
Circulating LH levels were significantly decreased
(P < 0.025) in rats treated with GH for 14 days via
the osmotic pumps relative to those in rats similarly treated with
saline (Fig. 1
). Treatment with GnRH
significantly increased (P < 0.001) plasma LH levels
in both groups of rats. However, the plasma LH response to GnRH
treatment in GH-treated rats was similar to that in GnRH-treated rats
previously implanted with saline-filled osmotic pumps. Basal and
GnRH-induced FSH secretion were similar in rats receiving saline and in
GH-treated rats (basal, 7.43 ± 0.14 vs. 7.66 ±
0.23 ng/ml; GnRH-treated, 9.27 ± 0.24 vs. 10.72
± 0.69 ng/ml). Administration of GH for 14 days resulted in a
significant (P < 0.01) decrease in plasma PRL levels
relative to those in control rats (Fig. 2). Plasma testosterone levels were
similar in rats treated with either saline or GH (data not shown).
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). As expected, treatment with TP
decreased LH secretion in both subgroups. However, the suppressive
effect of TP on LH secretion was significantly attenuated
(P < 0.025) in GH-treated rats on day 8 after
castration (day 14 after saline or GH treatment) relative to that in
similarly treated rats previously implanted with saline-filled osmotic
pumps (Fig. 3B). There were no significant differences in FSH responses
to castration and testosterone administration in these two subgroups of
rats (data not shown).
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and 5). Administration of GnRH significantly
increased (P < 0.001) plasma gonadotropin levels in
both subgroups of rats. However, the percent increase in plasma LH
levels after GnRH treatment was decreased in GH-immunized animals
(183% increase relative to 284% in adjuvant-injected controls).
Furthermore, the percent increase in circulating FSH levels was higher
in GH-immunized rats (165%) than in adjuvant-injected control rats
(139%). Plasma PRL levels were unaffected by GH immunization
(adjuvant-injected, 35.32 ± 2.3 ng/ml; GH-immunized, 30.32
± 2.1 ng/ml). Basal testosterone levels were similar in rats
previously injected with adjuvant and in rats immunized against GH. The
increased LH levels after GnRH treatment were associated with higher
(P < 0.05) plasma testosterone levels in
adjuvant-injected control rats, whereas the same treatment in
GH-immunized rats failed to alter testosterone secretion (Fig. 6).
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). However, in GH-immunized rats,
castration failed to increase plasma LH concentrations relative to
those in gonad-intact rats. As expected, administration of TP
significantly decreased (P < 0.001) plasma LH levels
in adjuvant-injected castrated rats. In contrast, TP treatment failed
to suppress plasma LH levels in GH-immunized castrated rats (Fig. 7B).
Although plasma FSH levels in GH-immunized castrated rats injected with
oil were higher than those in the corresponding adjuvant-injected
controls at each of the time periods examined, these apparent
differences were not statistically significant (Fig. 8A). The classical negative feedback
effect of TP on FSH secretion was significant (P <
0.001) in adjuvant-injected castrated rats, and similar treatment
failed to suppress plasma FSH levels in GH-immunized rats (Fig. 8, A
vs. B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it has been suggested that GH might play a role in the secretion of LH in the rat (8, 9, 10). Our previous studies have shown that the circulating LH levels were significantly increased in adult male transgenic mice expressing the human GH gene and in male GH-deficient Ames dwarf mice treated with bGH (7, 17). In the present study, in male rats, administration of GH for 14 days resulted in lower plasma LH levels, whereas active immunization against GH was associated with increased circulating LH levels. This suggests that there is a differential effect of GH with respect to LH secretion in rats and mice. Thus, GH tends to inhibit LH secretion in rats and to favor LH secretion in mice. As LH secretion is controlled primarily by GnRH, it is tempting to speculate that in rats, GH might have reduced the synthesis and/or release of GnRH as well as decreased the sensitivity of the pituitary gonadotrophs to GnRH action. This is corroborated by the present finding that plasma LH levels were decreased in GH-treated rats. The percent increase in plasma LH levels after GnRH treatment was decreased in GH-immunized rats. This indicates that the alterations in the normal GH milieu affect LH secretion and that physiological amounts of GH are required for the normal effects of GnRH. In contrast to the effect of GH on the circulating LH levels, GH treatment had no effect on either basal or GnRH-stimulated FSH secretion. Thus, the present data are consistent with previous reports (18, 19, 20) that the mechanisms that control FSH and LH secretion are different and that two separate releasing hormones might regulate the release of FSH and LH. Differential effects of GH on LH and FSH release could have also been due to altered frequency of GnRH pulses or changes in testicular secretion of steroid hormones and/or inhibin. It is also possible that monohormonal LH cells might exist in the pituitary gland and that these cells are the targets for GH action.
The castration-induced increase in LH secretion was decreased in GH-treated rats. In GH-immunized rats, there were no increases in plasma LH levels after castration. Also, the classical negative feedback effect of testosterone on gonadotropin secretion was impaired in rats immunized against GH. As expected, administration of TP decreased plasma gonadotropin levels in adjuvant-injected control rats, but the same treatment failed to reduce circulating LH and FSH levels in rats immunized against GH. This suggests that the sensitivity of the hypothalamic-pituitary system to the negative feedback effect of testosterone is reduced when the biological activity of the endogenous GH is neutralized. It is known that norepinephrine (NE) plays a critical role in GnRH secretion and that alterations in NE turnover affect LH secretion (21, 22). In the present study, the attenuated effect of testosterone on LH secretion in GH-immunized rats might have been due to higher hypothalamic NE turnover and the inability of testosterone to alter NE synthesis.
In the present study, rats treated with GH for 14 days became hypoprolactinemic, indicating that GH modulates the synthesis and/or release of PRL by the pituitary acidophils. It is known that a decrease in PRL secretion can usually be related to the increase in median eminence dopamine turnover (23, 24, 25). Therefore, it is reasonable to infer that the hypoprolactinemia in GH-injected rats might have been due to an alteration in dopamine turnover. It is known that bGH is largely somatotropic in function (26), yet it altered PRL secretion. In our previous study in transgenic mice, expression of the bGH gene with mouse metallothionein I promoter induced mild hyperprolactinemia (27). It has been shown that expression of the foreign gene with mouse metallothionein I promoter starts during fetal development and continues throughout the lifespan (28, 29). Therefore, the results of these two distinctly different studies indicate that the effect of GH on PRL secretion is variable and apparently depends on the species studied and/or the length of the exposure of the neuroendocrine system to GH. It is interesting to note that the circulating PRL levels were not affected in rats immunized against GH, implying that only excess GH can activate the tuberoinfundibular dopaminergic neurons and affect PRL secretion.
In rats, administration of PRL has been shown to increase dopamine turnover in the terminals of the tuberoinfundibular neurons (25, 30) and decrease endogenous PRL secretion (31, 32). The bGH preparation used in the present study was provided by the USDA, and it contained 4–10% bPRL as a contaminant. Therefore, it is possible that the PRL impurity within the bGH preparation might have activated the tuberoinfundibular dopaminergic system and, in turn, induced hypoprolactinemia in rats treated with this GH. In addition, the decrease in plasma LH levels in our study might have been due to the PRL impurity, because it is known that hyperprolactinemia reduces LH secretion in rats (33, 34, 35). However, in these studies, large amounts of PRL (400 µg/100 g BW/injection, three times a day) have been used to induce hyperprolactinemia. We have treated rats with small amounts of GH, and the bPRL content was relatively low. Therefore, in our study it is very unlikely that the bPRL contaminant influenced the circulating PRL and LH levels.
The increased LH secretion after GnRH treatment resulted in higher plasma testosterone levels in adjuvant-injected animals. However, similar treatment in GH-immunized rats failed to increase circulating testosterone concentrations. Also, the higher basal LH levels observed in GH-immunized rats did not alter testosterone secretion. It has been shown that GH increases LH receptors of the testes and enhances the efficacy of gonadotropin in testosterone secretion in rats (4, 5, 36). Therefore, in the present study, biological neutralization of endogenous GH might have affected the action of LH on Leydig cell steroidogenesis. It is possible that GH immunization might have affected the Leydig cells and, therefore, modified the LH action. It is known that IGF-I is produced by the liver and that its secretion is controlled by GH (37, 38). Leydig cells have IGF-I receptors, and IGF-I has been shown to modulate the effect of gonadotropin on testosterone secretion by the isolated Leydig cells (39, 40, 41). In the present study, plasma IGF-I levels were reduced in GH-immunized rats. Therefore, it is suspected that a reduction of IGF-I secretion might have been an added influence on decreased testosterone responses in these rats.
The effects of exogenously administered bGH on LH and PRL secretion can be related to the effects of GH because treatment with bGH did not result in the development of antibodies against GH, and circulating bGH was detected in animals infused with bGH. In the present study, a modest decrease in plasma IGF-I levels in bGH-treated animals is surprising and is possibly due to a negative feedback effect of IGF-I at the time blood samples were collected. In the human, there are precedents for GH treatment failing to increase IGF-I secretion (42). Therefore, it is most likely that the dosage, mode of administration, and/or duration of GH treatment might have an effect on IGF-I secretion. Furthermore, it has been shown in the rat that administration of GH increased hypothalamic somatostatin levels (43), and treatment with a somatostatin analog increased serum IGF-I levels (44), suggesting that somatostatin might influence IGF-I secretion. Therefore, in the present experiment, it is possible that GH infusion might have affected the release of somatostatin and influenced the secretion of IGF-I.
In summary, the findings reported here demonstrate that induction of GH excess by GH treatment induced hypoprolactinemia and decreased LH secretion, whereas biological neutralization of endogenous GH increased circulating gonadotropin concentrations and altered the effects of GnRH as well as testosterone on LH secretion. Increased basal and GnRH-stimulated LH secretion did not affect Leydig cell function in GH-immunized rats. Thus, GH modulates hypothalamic-pituitary-testicular function in adult male rats.
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Acknowledgments |
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Footnotes |
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Received June 4, 1997.
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