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Pituitary and Testicular Function in Growth Hormone Receptor Gene Knockout Mice¹

Department of Physiology, Southern Illinois University School of Medicine (V.C., A.B.), Carbondale, Illinois 62901-6512; and Edison Biotechnology Institute, Molecular and Cellular Biology Program, Ohio University (K.T.C., J.J.K.), Athens, Ohio 45701

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.

	Abstract

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The role of GH in the control of pituitary and testicular function is poorly understood. GH receptor gene knockout (GHR-KO) mice were recently produced. As these mice are good experimental animals to assess the influence of the effects of GH and insulin-like growth factor-I (IGF-I), the present studies were undertaken. Young adult male GHR-KO mice and their normal siblings were tested for fertility and subsequently injected (ip) with saline or GnRH (1 ng/g BW) in saline. Fifteen minutes later, blood was obtained via heart puncture. Plasma IGF-I, PRL, LH, and testosterone concentrations were measured by RIAs. In addition, the testicular testosterone response to LH treatment was evaluated in vitro. The results indicate that the absence of GH receptors (GHRs) was associated with an increase (P < 0.005) in plasma PRL levels, and circulating IGF-I was not detectable. Although the basal plasma LH levels were similar in GHR-KO mice relative to those in their normal siblings, the circulating LH response to GnRH treatment was significantly (P < 0.001) attenuated. Plasma testosterone levels were unaffected by disruption of the GHR gene. However, basal (P < 0.01) and LH-stimulated (P < 0.001) testosterone release from the isolated testes of GHR-KO mice were decreased. The rate of fertility in GHR-KO male mice was also reduced. These results indicate that the lack of GHRs (with GH resistance and lack of IGF-I secretion) induces hyperprolactinemia and alters the effect of GnRH on LH secretion as well as testicular function. Thus, GH and IGF-I influence pituitary and gonadal functions in male mice.

	Introduction

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THE EFFECTS of GH on growth and metabolism have been known for many years, but its role in the control of neuroendocrine and gonadal function in mammals is poorly understood. However, studies have suggested that GH may be involved in ovarian function (1, 2). GH antigens are identified in pituitary cells containing FSH or LH messenger RNAs and in GnRH receptors, indicating that either GH cells are transitory gonadotrophs, or GH is present in these pituitary cells, possibly to control their function (3, 4). Furthermore, GH-binding protein antigens were identified in pituitary cells that contained LH and FSH, indicating a paracrine effect of GH on the function of the gonadotrophs (5). Thus, GH may function as a "cogonadotropin" (3, 4). It has been shown that GH administration to oligospermic men enhances the efficacy of exogenous gonadotropins in the induction of sperm production (6). In men, congenital GH resistance due to mutated GH receptors (Laron syndrome) is associated with a delay in sexual maturation (7, 8), suggesting that GH exerts an important function in reproduction. In addition, results from experimental animals have suggested that GH may play an important role in the control of pituitary and testicular functions. It has been shown that GH treatment of hypophysectomized rats, increases the LH receptor content of the testis (9) and enhances the testicular responsiveness to gonadotropin treatment (10). In adult rats, a lack of GH secretion results in a delay in testicular growth and differentiation of germinal cells (11). In our recent study, we have shown that treatment with GH or biological neutralization of endogenous GH by active immunization against GH alters gonadotropin secretion in adult male rats (12). In addition, we have previously shown that administration of GH to GH-deficient Ames dwarf mice increases plasma LH levels (13), indicating that GH might be involved in the control of gonadotropin secretion. However, Ames dwarf mice are also deficient in PRL and TSH (14, 15) and, therefore, are a less suitable model to study the effects of GH. Recently, GH receptor gene knockout (GHR-KO) mice were produced (16). Although these mice secrete large amounts of GH (16), it is assumed that the effects of the secreted GH are absent due to the lack of GH receptors (GHRs). Therefore, these GHR-KO mice are good experimental animals to test the role of GH in neuroendocrine and gonadal function. In this report we have attempted to identify the alterations in fertility, pituitary, and gonadal function in male GHR-KO mice relative to those in their normal siblings.

	Materials and Methods

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Genotype determination of the mice used in the present experiments
The genotype of each mouse was determined by PCR analysis of its genomic DNA using a modified version of the procedure described by Eisen et al. (17). In short, an approximately 0.5-cm piece of tail was digested with 400 µl 1 mg/ml proteinase K in SSTE (1% SDS, 100 mM NaCl, 50 mM Tris, and 15 mM EDTA, pH 8) overnight at 55 C. RNA was then degraded with ribonuclease A, protein was removed by phenol-chloroform-isoamyl alcohol (25:24:1) extraction, and DNA was precipitated with sodium acetate and ethanol and redissolved in 300 µl water. PCR was performed on the genomic DNA samples using a combination of three primers: In3+1 (5'-CCTCCCAGAGAGACTGGCTT-3'), In4–1 (5'-CCCTGAGACCTCCTCAGTTC-3'), and Neo-3 (5'-GCTCGACATTGGGTGGAAACAT-3'). Each PCR sample [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2 mM MgCl₂, 0.2 mM deoxy (d)-ATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 0.5 µM of each primer, 0.05 U Taq DNA polymerase, and 0.13 µl genomic DNA/µl reaction] was amplified in a Perkin-Elmer GeneAmp 9600 (1 cycle of 95 C for 2 min followed by 30 cycles of 95 C for 15 sec, 58 C for 20 sec, and 72 C for 30 sec) and then electrophoresed through a 1% agarose, 1% Metaphor agarose (FMC, Rockland, ME), 1 x TAE (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA), 0.2 µg/ml ethidium bromide gel. Normal siblings produced a single fragment of about 390 bp, homozygous animals produced two fragments of approximately 220 and 290 bp, and heterozygous animals produced all three fragments.

In our laboratory, we found that there were no differences in body weight, basal plasma LH levels, or the LH response to GnRH treatment between homozygous (+/+) and heterozygous (+/-) normal mice. It has been also reported that these two genotypes are not significantly different in terms of secretion of GH, insulin-like growth factor-I (IGF-I), and GHR/GH-binding protein (16). Therefore, in the present studies data from +/+ and +/- mice were combined for statistical analyses and reported as normal mice.

Animals
GHR-KO mice (-/-) were produced as described previously (16). Adult normal female mice bred in our animal facility were mated with either GHR-KO male mice or male mice with the +/- genotype, and the resulting male GHR-KO mice and non-GHR-KO littermates (normal mice) were used in the present experiments. Mice were housed in a room with a controlled photoperiod of 12 h of light/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, PMI Feeds, Inc., St. Louis, MO) and tap water. The following experiments were conducted.

Fertility testing
Each GHR-KO or normal male mouse was housed with two young adult virgin females for 10 days immediately preceding the experiments described below. The females were checked daily for birth of litters, and the numbers of live and dead pups were recorded.

In vivo experiment.Male GHR-KO mice and their normal siblings (12–13 weeks of age) were divided into two groups (n = 9–10 mice/group) and treated ip as follows: group 1, saline; and group 2, GnRH in saline (1 ng/g BW; Sigma Chemical Co., St. Louis, MO; lot 106F-58302). We have shown previously that this dose of GnRH is effective in inducing LH secretion in normal mice (13, 18). Fifteen minutes after saline or GnRH injection, blood was obtained via heart puncture under ether anesthesia. Plasma samples were frozen at -20 C until assayed for LH, PRL, IGF-I, and testosterone. The pituitary, testes, and male accessory reproductive organs (seminal vesicles and ventral prostate) were removed and weighed with their secretions. Testes from saline-injected mice were removed and used in the following in vitro experiment.

In vitro experiment. Testes were decapsulated and weighed. The decapsulated testes were cut into small fragments of approximately equal weights. One testicular fragment was used per incubation. Testicular incubations were performed as described previously (13, 19). Briefly, testicular fragments were preincubated in Krebs-Ringer bicarbonate buffer (2 ml) containing 1% glucose for 30 min in a Dubnoff metabolic incubator in an atmosphere of 95% oxygen-5% carbon dioxide at 32 ± 2 C with constant shaking at 100 rpm. At 30 min, the incubation medium was removed and replaced with the same buffer (1.9 ml) containing glucose. In addition, either 0.1 ml saline or 5 ng ovine LH (NIH LH-26) in 0.1 ml saline were added, and incubation was continued for 4 h. Incubations were terminated at 4 h, and the medium was used to determine testosterone levels by RIA.

Hormone assays
The concentrations of LH in plasma were determined by RIA as described previously (13, 18), using reagents generously supplied by Dr. A. F. Parlow, Dr. G. D. Niswender, and the National Hormone and Pituitary Program, NIH (Bethesda, MD). Briefly, rat LH RP-2 reference preparation and ovine LH antiserum (GDN-15) were used in the LH RIA. Various amounts of a plasma pool obtained from intact and castrated mice produced curves parallel to those of various amounts of rat LH reference preparation. Therefore, it is valid to use these reagents to measure LH levels in mice. The sensitivity of this assay was 10 pg/tube. All plasma samples were measured starting on the same day, using the same day diluted reference preparation, antiserum, and repurified hormone trace. The intraassay coefficient of variation was 2.8%.

The plasma PRL concentrations were measured by RIA as we previously described (18). Briefly, mouse PRL reference preparation (AFP-6476C) and mouse PRL antiserum (AFP-131078; both provided by Dr. A. F. Parlow) 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 4.3%.

Plasma testosterone levels were determined by RIA as described previously (12, 13, 18) with a standard extraction (extracted with the anhydrous diethyl ether) procedure. The sensitivity of this testosterone assay was 5 pg/tube. The mean intraassay coefficient of variation was 2.9%.

Plasma IGF-I concentrations were measured by RIA as described by us and others (13, 20, 21). 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 (20). Because this extraction method does not eliminate all IGF-I-binding proteins present in the plasma (21), acid-acetone extracts were subjected to cryoprecipitation, a procedure described previously (21). The mean recoveries of iodinated IGF-I added to the plasma were 90.5%. 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, Inc. (Thousand Oaks, CA) was used as the reference preparation, and human IGF-I (A52–8MH-144; Eli Lilly & Co., Indianapolis, IN) was iodinated and used as 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, 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. Therefore, it is valid to use these human IGF-I RIA reagents to measure IGF-I levels in mouse plasma. The sensitivity of this assay was 32 pg/tube. All plasma extracts were included in the same assay to avoid interassay variability. The intraassay coefficient of variation was 2.4%.

Statistical analysis
Statistical analyses were performed by ANOVA followed by the Student-Newman-Keuls test. Student’s t test was used when the values of two groups were compared. Data on the incidence of male fertility were analyzed by a ² test.

	Results

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Body, pituitary, and accessory reproductive organ weights
As expected, the mean body weight of GHR-KO mice was significantly (P < 0.001) lower (12.0 ± 0.4 g) than that of normal controls (26.8 ± 0.4 g). Similarly, there were dramatic (P < 0.001) decreases in the pituitary (0.91 ± 0.06 vs. 1.71 ± 0.07 mg), testicular (111.3 ± 6.4 vs. 211.0 ± 8.3 mg), seminal vesicle (92.5 ± 6.0 vs. 187.1 ± 11.7 mg), and ventral prostate (5.9 ± 0.4 vs. 9.8 ± 0.5 mg) absolute weights in GHR-KO mice relative to those in normal mice.

Fertility
Of 22 normal males, 21 impregnated at least 1 of the normal females with which they were housed and thus were considered fertile. However, only 14 of 19 GHR-KO males were fertile by the same criteria (P < 0.05). As each male was mated with 2 females, we also calculated the percentage of females that became pregnant in each of the groups. Eighty-eight percent of the females housed with normal males and 53% of the females housed with GHR-KO males became pregnant (P < 0.001). Litter size, defined as a mean number of live pups, did not differ between litters sired by normal and GHR-KO males (6.4 ± 0.4 vs. 5.9 ± 0.4 pups). There were also no significant differences in the mean numbers of pups found dead in the 2 groups.

Plasma IGF-I and PRL concentrations
Plasma IGF-I levels were 164.4 ± 8.6 ng/ml in normal siblings, and IGF-I was not detectable in the circulation in GHR-KO mice.

Circulating PRL concentrations were significantly (P < 0.005) increased in GHR-KO mice relative to those in their normal siblings (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Figure 1. Circulating PRL levels in normal and GHR-KO mice. Values are means. Vertical lines represent the SEM. Values without the same letter are at a significance level of at least P < 0.05.

View larger version (14K): [in this window] [in a new window]   Figure 2. Circulating LH levels in normal and GHR-KO mice given either saline or GnRH in saline. Values are means. Vertical lines represent the SEM. Values without the same letter are at a significance level of at least P < 0.05.

View larger version (15K): [in this window] [in a new window]   Figure 3. Circulating testosterone levels in normal and GHR-KO mice given either saline or GnRH in saline. Values are means. Vertical lines represent the SEM. Values without the same letter are at a significance level of at least P < 0.05.

View larger version (15K): [in this window] [in a new window]   Figure 4. In vitro testosterone production (per mg testes). Testicular fragments were obtained from normal or GHR-KO mice and exposed to saline or 5 ng LH in saline. Vertical lines represent the SEM. Values without the same letter are at a significance level of at least P < 0.05.

View larger version (13K): [in this window] [in a new window]   Figure 5. In vitro total testosterone production (per two testes). Testicular fragments were obtained from normal or GHR-KO mice and exposed to saline or 5 ng LH in saline. Vertical lines represent the SEM. Values without the same letter are at a significance level of at least P < 0.05.

	Discussion

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Delayed sexual maturation in female GHR-KO mice has been reported, and mating of GHR-KO females with GHR-KO males resulted in reduced litter size (16). In the present study there was a reduction in the fertility of GHR-KO male mice, possibly due to hyperprolactinemia. In the male mouse, hyperprolactinemia was reported to stimulate sexual behavior (22), and we are not aware of any studies of fertility in hyperprolactinemic male mice. However, it has been shown that hyperprolactinemia results in a slight reduction in fertility in female mice (23). The basal testosterone production in vitro and its response to LH treatment were reduced in GHR-KO mice. As testosterone plays an important role in spermatogenesis, it is possible that sperm production is affected by disruption of the GHR gene, and this may have contributed to reduced fertility in these male mice. Subfertility in little (lit/lit) mice with isolated GH deficiency has been shown to be due to longer mount, intromission, and ejaculation latencies (24). Therefore, alterations in sexual behavior might have affected fertility in GHR-KO mice.

It has been shown that GH secretion is increased (16), and in the present study the circulating IGF-I levels were not detectable in GHR-KO mice. We and others have shown in transgenic mice that expression of the various GH or IGF-I genes is associated with accelerated body growth (18, 25, 26). Induction of hyperprolactinemia increases seminal vesicle weight in mice (27). However, in the present study, despite increased PRL secretion, there were decreases in seminal vesicle and ventral prostate weights in GHR-KO mice. In transgenic mice expressing various GH genes, seminal vesicle weights are increased (28), possibly due to increases in IGF-I secretion (29). A significant reduction in BW and in the weight of accessory reproductive structures of GHR-KO mice is most likely due to the impairment of IGF-I secretion and resistance to the endogenously secreted GH.

Functional deficits of the pituitary-testicular axis and the reduced growth rate in GHR-KO mice could be related. It is known that gonadal steroids can influence growth rate and that experimental alterations in testosterone secretion could change the pattern of GH secretion and consequently affect growth (30). In rats, gonadal steroids may affect growth indirectly by altering the effectiveness of GH by modulating the hepatic GH receptor levels (31). In GHR-KO mice, the absence of GHRs and IGF-I secretion was associated with reduced body weight and testicular function. It would appear that the endogenously secreted testosterone is relatively ineffective in inducing growth in GHR-KO mice. This hypothesis is supported by the finding that testosterone treatment does not stimulate growth in hypophysectomized rats (32). Hence, the reduced body weight is probably not due to the alterations in the pituitary-testicular function in GHR-KO mice, but is most likely due to the absence of GHRs and IGF-I secretion. However, it remains to be determined whether the reduced growth rate and body size are involved in mediating the effects of GH resistance on the neuroendocrine-testicular axis in GHR-KO mice.

The present study is the first to demonstrate that GHR-KO mice are hyperprolactinemic. Our previous study has shown that in transgenic mice expressing the bovine GH gene, PRL secretion is increased (33), and dopamine turnover in the median eminence of these animals is reduced (34). As GHR-KO mice produce large amounts of GH (16), it is conceivable that GH might have influenced the function of the pituitary lactotrophs. It is known that dopamine suppresses PRL secretion (35, 36). Therefore, it is tempting to speculate that the excess GH produced in GHR-KO mice might have suppressed tuberoinfundibular dopaminergic neurons, resulting in increased PRL secretion. However, these animals lack GHRs (16). Therefore, we speculate that the mode of GH action in increasing PRL secretion may have been via a different and unknown mechanism. It has been shown that experimental induction of hyperprolactinemia in adult male DBA/2J mice is associated with increase in circulating LH levels (37, 38). However, in GHR-KO mice, despite the hyperprolactinemic condition, basal plasma LH levels were not affected. This might have been due to either the lack of a direct effect of GH on LH-secreting pituitary cells or the absence of IGF-I secretion. As in mice both excess GH production and GH deficiency are associated with altered PRL secretion (14, 15, 18, 33), it may be not possible to separate direct from PRL-mediated effects of GH in the in vivo situation.

Although basal plasma LH levels were not affected, the LH response to GnRH treatment was significantly decreased in GHR-KO mice. This indicates that in these animals, the GnRH action may be affected due to the absence of IGF-I secretion and that GH by itself is unable to restore the normal effect of GnRH on LH secretion. We have shown in GH-deficient Ames dwarf male mice that induction of IGF-I secretion by GH administration improves the LH response to GnRH treatment (13). The attenuated LH response to GnRH treatment in GHR-KO mice is possibly also due to hyper-prolactinemia. It has been shown that hyperprolactinemia attenuates the effects of GnRH in the rat (39, 40) and suppresses the priming effect of GnRH on LH secretion in normal and hypogonadal mice (41).

The lack of GHRs in GHR-KO mice did not affect basal testosterone secretion, possibly due to the normal amounts of LH secretion. Furthermore, the testes of GHR-KO mice responded to the increased secretion of LH after GnRH administration and increased plasma testosterone concentrations. However, this testosterone response was attenuated in GHR gene-disrupted mice relative to that in normal mice. In addition, basal testosterone secretion by the isolated testis was attenuated in GHR-KO mice. The in vitro study also indicated that the testosterone response to LH treatment by the testes of GHR-KO animals was significantly decreased. These data suggest that the effect of LH is influenced, possibly by the subnormal function of the Leydig cells of the testes of GHR-KO mice. GH treatment to hypophysectomized male rats has been shown to increase the LH receptor content of the testis (9), and induction of hyperprolactinemia in mice decreases testicular hCG-binding sites (37), suggesting that GH deficiency and hyperprolactinemic condition in GHR-KO mice might have affected LH receptor number and their function within the testes. It has also been demonstrated that Leydig and Sertoli cells contain IGF-I receptors and that IGF-I can modulate the effects of LH on testosterone secretion by the isolated Leydig cells (42, 43, 44). Therefore, it is possible that due to the lack of production of IGF-I, the full effect of LH on Leydig cells is attenuated in GHR-KO mice.

It has been shown that rat testis contains IGF-I messenger RNA (45, 46). In addition, cultured Sertoli cells and Leydig cells from adult rats secrete IGF-I (47). Treatment of Sertoli cells with FSH and of Leydig cells with LH resulted in increases in IGF-I levels in culture medium (48), suggesting that the secretion of IGF-I might not be dependent on GH. Therefore, it is possible that testicular IGF-I secretion might be normal in GHR-KO mice. However, in the present study the attenuated testosterone response to LH suggests that extratesticular IGF-I may play an important role in the action of LH on testosterone secretion by the testis.

There are some similarities in reproductive characteristics of male GHR-KO mice and those of IGF-I gene-disrupted mice. Similar to GHR-KO mice, the weights of male sex accessory structures were reduced in targeted IGF-I gene-disrupted mice (49). In addition, their circulating testosterone levels were reduced. As in the present study, the in vitro testosterone response to LH treatment was suppressed in the absence of the IGF-I gene (49). However, the IGF-I gene-disrupted mice were infertile. In contrast, fertility is reduced, but not totally suppressed, in GHR-KO mice. The mechanism responsible for the maintenance of fertility in GHR-KO mice is unknown. Furthermore, GH treatment increased IGF-I secretion and consequently elevated the total number of viable spermatozoa in GH-deficient dwarf rats (50) and led to an absence of mating behavior in mice with IGF-I gene null mutation (49), strongly suggesting that IGF-I is important for normal male reproduction.

In conclusion, the present data indicate that targeted disruption of the GHR gene influences the GnRH effect on LH secretion by the pituitary gland, induces hyperprolactinemia, attenuates testicular endocrine function, and reduces fertility in GHR-KO mice. Some of the effects observed in GHR-KO mice might have been due to the lack of IGF-I secretion.

	Acknowledgments

 
We thank Dr. G. D. Niswender, Colorado State University (Fort Collins, CO); Dr. A. F. Parlow, Pituitary Hormone and Antisera Center, Harbor-University of California-Los Angeles Medical Center (Torrance, CA); and the National Hormone and Pituitary Program (Rockville, MD) for generously providing reagents used in testosterone, IGF-I, and pituitary hormone RIAs. Dr. R. B. Bowsher, Eli Lilly & Co. Laboratory for Clinical Research and Eli Lilly & Co. (Indianapolis, IN) generously supplied the recombinant human IGF-I used in the IGF-I RIA.

	Footnotes

 
¹ This investigation was supported by NIH Grant HD-20001.

Received July 16, 1998.

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