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Department of Physiology, Southern Illinois University School of Medicine (V.C., A.B., L.D.R.), Carbondale, Illinois 62901-6512; Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center (C.A.A.), Denver, Colorado 80262; Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development (C.H.T.-M., M.L.D.), National Institutes of Health, Bethesda, Maryland 20892-4510; and Edison Biotechnology Institute and Department of Biomedical Sciences, College of Osteopathic Medicine (J.J.K.), Ohio University, 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.
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Abstract |
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Introduction |
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It is known that the majority of GH effects are due to the action of IGF-I. GH acts on the hepatic cells and initiates the synthesis and secretion of IGF-I. It has 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 (7, 8, 9). We have shown previously that circulating IGF-I is undetectable in GH-deficient Ames dwarf mice and exogenous GH administration induces IGF-I secretion (10). However, these mice are also TSH and PRL deficient (11, 12). Furthermore, it has been reported that some of IGF-I gene disrupted mice die shortly after birth (13). Thus, lack of a suitable animal model has become a limiting factor to assess the effects of GH/IGF-I on reproductive function. In our previous study (14), we have assayed circulating IGF-I levels in male GH receptor gene knockout (GHR-KO) mice and in their normal siblings after subjecting the plasma samples to the two-step procedure to remove IGF-I binding proteins, which are known to interfere with IGF-I measurements (15, 16). Unlike in normal mice, GHR-KO mice have no detectable IGF-I in circulation (14). Therefore, GHR-KO mice are good experimental models to study the role of IGF-I in reproductive function and in the present study we have evaluated the endocrine function of the testes of these mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments and blood collection
Each group of adult (12–16 weeks of age) male GHR-KO mice and
their normal siblings was divided in to two subgroups (n = 7–9
mice/subgroup) and treated (single ip injection) with either saline or
ovine LH (oLH) (NIH-26; 0.3 µg/g BW) in saline. Our previous study
has shown that this dose of LH was effective in increasing the
circulating androgen levels in mice (18). One hour after
saline or LH administration, blood was obtained via heart puncture
under isoflurane anesthesia. Plasma samples were stored at -20 C until
assayed for IGF-I, LH, FSH, PRL, androstenedione, and testosterone
levels by RIAs. The testes were removed, weighed, immediately frozen on
dry ice and kept in -60 C until assayed for LH and PRL receptors.
Testicular LH and PRL receptor assays
Hormone preparations. Purified human CG (hCG; CR119, Center
for Population Research, NIHCD) and human GH (hGH; HS 2243) were used
in these receptor assays.
Radioidodination of hCG and hGH and radioreceptor assays. 125I-hCG and 125I-hGH (specific activity, 40–60 µCi/µg) preparations were obtained by radioiodination using the lactoperoxidase method (19). The radioiodinated hormones were purified by chromatography on Sepharose-Concanavalin A (20) and Sephadex G-100 (21), respectively. The maximal bindability of the tracer preparations ranged from 50–70%.
Measurements of LH and lactogen binding to testicular homogenates
of GHR-KO and normal mice. Frozen testes, one from each of GHR-KO
mice (n = 9) and normal siblings (n = 9) were individually
homogenized in Dulbecco’s PBS (pH 7.4) in a glass homogenizer. Protein
concentration was determined by protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA).
125I-binding of hGH and hCG assays in homogenized
testes (100 µg/50 µl·tube) were performed as previously described
(21, 22) in the presence of near saturating concentration
of hGH or hCG (5 ng) in a total assay volume of 250 µl. Nonspecific
binding was determined in samples containing an excess of unlabeled
hormone (1 µg). Samples were incubated for 16 h at 22 C.
Incubations were terminated by the addition of ice-cold 0.1% BSA in
PBS. The receptor bound fraction was separated from the free hormone by
centrifugation and counted in an automatic 
-scintillation counter.
All assays were performed in triplicates.
RNA extraction, Northern blots, and probes used for
hybridization
To evaluate the possible effects of GHR gene disruption on
pituitary and Sertoli cell functions, pituitaries as well as testes of
GHR-KO and normal mice were used to assess the expression of
LHß-subunit in pooled pituitaries (four pituitaries per RNA
extraction) and sulfated glycoprotein-2 (SGP-2) mRNA in testes using
conditions described previously (23, 24, 25). Briefly, total
RNA was isolated from pituitaries and testes as described by
Chomczynski and Sacchi (26). Aliquots of RNA (5 µg;
n = 3–4/group) were electrophoresed through denaturing 1.2%
agarose gel and subsequently blotted by capillary action to nylon
filters. The filters with pituitary RNA were hybridized with LHß
probe (kindly provided by Dr. Joseph L. Roberts, Mt. Sinai School of
Medicine, New York, NY) and filters containing testicular RNA were
hybridized with SPG-2 probe (kindly supplied by Dr. Michel D. Griswold,
Washington University, Pullman, WA). The relative amount of the gene
transcripts was determined by densitometer (Bio-Rad Laboratories, Inc. Model GS-670). The data were corrected for differences in
gel loading using the 18s ribosomal RNA signals as standards as
described in our previous publication (25).
Hormone assays
Plasma IGF-I levels were measured by RIA as described by us and
others (10, 15, 16). Because 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 (10, 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 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. Biologicals (Thousand Oaks, CA) was used as the reference
preparation, and the human IGF-I (No. A52–8MH-144; Eli Lilly & 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. 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
3.1%.
The concentrations of LH and FSH in plasma were determined by RIAs as described previously (10, 27) using reagents generously supplied by Dr. A. F. Parlow, Dr. G. D. Niswender, and National Hormone and Pituitary Program, NIH. Briefly, rat (r) LH RP-2 reference preparation, oLH antiserum (GDN-15), rFSH RP-2 reference preparation, rFSH antiserum (S-11) were used in LH and FSH RIAs, respectively. Various amounts of plasma pools obtained from intact and castrated mice produced curves parallel to those of varied amounts of rat LH and rFSH reference preparations. Therefore, it is valid to use these reagents to measure 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 were measured starting on the same day, using the same day diluted reference preparation, antiserum, and repurified hormone trace. The intraassay coefficients of variation was 5.1% for LH and 4.6% for FSH.
The plasma PRL concentrations were measured by RIA as previously described by us (27). Briefly, mouse PRL reference preparation (AFP-6476C) and mouse PRL antiserum (AFP-131078, both kindly 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 3.6%.
Plasma androstenedione and testosterone levels were determined by RIAs as described previously (18, 28) with a standard extraction (extracted with anhydrous diethyl ether) procedure. The androstenedione antibody (X-322) used to measure plasma androstenedione levels was purchased from the Southwest Foundation for Biomedical Research (San 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 sensitivity 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 variations were 2.6% for androstenedione and 2.3% for testosterone.
Testicular morphometry
Tissue preparation. Fifteen minutes before administration of
anesthesia with Nembutal (5 mg/100g BW), mice (normal and GHR-KO mice,
n = 4 mice/group) were perfusion fixed as previously described
(29). Testes were postfixed, dehydrated,and embedded in
plastic, sectioned (1 µm) stained in toluidine blue. After fixation,
the testis weights were averaged and each testis was used equally in
the following determinations:
Morphometry and measurements
Basic stereological principles were employed (30, 31). The volume density of testicular lumen, interstitium and
tubule were assessed by point counting morphometry at x100 using a
441-point lattice. The data were provided as a percentage by
multiplying times 100. Over 4,000 points were scored for each testis
examined.
Fifty tubular diameters for each testis were measured using an ocular measuring device. The tubular diameter was measured in 50 round or nearly round seminiferous tubule profiles from each testis at x100 magnification. The shortest dimension of each tubule was measured. The ocular micrometer was calibrated with a stage micrometer to yield the final measurement.
The seminiferous tubule length was assessed by using the following
formula:
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Point counting morphometry was used to measure the volume density of the Leydig and Sertoli cells in each testis examined at x450 magnification. Over 10,000 points were scored and data were provided as percentage by multiplying times 100. The concentrations of Leydig and Sertoli cells were expressed as µg/mg testis as well as µg/testis. For this purpose, the total volume of the Leydig and Sertoli cell nucleus were calculated as: the product of the volume density and testis weights then calculated for mg and total testis weight.
Statistical analyses
Statistical analyses were performed by ANOVA followed by the
Student’s-Neuman-Keuls test. t test was used when values of
two groups were compared. Testicular LH and PRL receptor data were
analyzed for statistical differences by ANOVA followed by Duncan’s
multiple range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). The number of
testicular PRL receptors, assessed by 125I-hGH
binding in the testicular homogenates was also significantly reduced
(fmol/mg protein: P < 0.005; fmol/testis:
P < 0.001) in GHR-KO mice relative to the normal mice
(Table 1). The testicular LH and PRL receptors expressed as fmol/µg
Leydig cell volume per testis were also significantly reduced (LH
receptor, P < 0.001; PRL receptor, P
< 0.002) in GHR gene disrupted mice.
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. The levels of
pituitary LHß subunit mRNA in male GHR-KO mice were similar to those
of normal siblings. Similarly, testicular SGP-2 mRNA levels in GHR gene
disrupted mice were not significantly different than in normal control
mice (Fig. 2).
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). Plasma PRL levels
were higher (P < 0.01) in GHR-KO mice than in their
normal siblings (Fig. 3B).
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). This response to LH treatment was
not significantly different in these two groups of mice.
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).
Testicular morphometry
The length of the seminiferous tubules of GHR-KO mice was greatly
reduced. The diameter of the seminiferous tubules was significantly
reduced in GHR gene disrupted mice relative to the normal mice (Table 2). Although the testicular weights were
reduced by approximately 50%, the lumen, interstitium, and
seminiferous epithelium (tubule) were almost proportional in normal and
GHR-KO mice. However, there was a statistically significant increase in
the percent volume density of seminiferous epithelium and a significant
decrease in testicular lumen in GHR-KO mice.
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Discussion |
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Disruption of GHR gene resulted in decreased body weight relative to their normal siblings. Although GHR-KO mice secrete large amounts of GH (17), due to the absence of GHRs, IGF-I is not secreted which results in reduced body growth. The average testicular weights of GHR-KO mice were also reduced as in our previous studies (14, 17), suggesting that for normal growth of the testes, physiological amounts of GH, GHRs and, IGF-I are required. This finding is consistent with the previous study demonstrating a delay in testicular growth in rats lacking GH secretion (34) or treated with antiserum to GHRH (6).
In the present study, the basal secretion of LH was not affected by GHR gene disruption, consistent with normal LHß mRNA levels in pituitary glands of these mice. However, the testosterone response to LH treatment was significantly attenuated in GHR-KO mice. Absence of GHRs and IGF-I secretion in GHR-KO mice was associated with a significant decrease in the testicular LH receptors. Presumably, due to the reduction in the number of testicular LH receptors, the action of the exogenously administered LH on Leydig cells is attenuated in GHR gene disrupted mice. It has been shown that GH treatment to hypophysectomized rats enhance the LH receptor contents of the testes (4), supporting the concept that GH participates in the maintenance of LH receptor numbers and the function of the testis. In GH-, PRL-, and TSH-deficient Snell dwarf mice, administration of IGF-I increased testicular LH receptors and steroidogenic response (35). Zhang et al. (36) have demonstrated that IGF-I up-regulates LH receptor mRNA and binding sites in cultured murine Leydig cells. In male mice carrying a null mutation of the IGF-I gene, the weights of reproductive organs are reduced and these mice are infertile (37). The volume and number of Leydig cells are drastically reduced in these mice. These previous studies and results of the present investigation suggest that IGF-I plays an important role in the regulation of testicular function.
Mice with disrupted GHR gene are hyperprolactinemic. Our previous study has shown an increase in PRL secretion, and a reduction in median eminence dopamine turnover in transgenic mice expressing the bovine GH gene (38, 39). Although GHR-KO mice secrete GH (17), because of the absence of GHRs, it might be inferred that the secreted GH has no effect on dopamine turnover to influence the PRL synthesis and release. Therefore, stimulation of PRL secretion in GHR-KO mice may be through a different and unknown mechanism. Experimentally induced hyperprolactinemia in male DBA/2J mice resulted in elevation of circulating LH levels associated with an increase in norepinephrine turnover in the median eminence (40, 41). In contrast, despite the fact that GHR-KO mice are hyperprolactinemic, their basal plasma LH levels were similar to those in normal siblings. Furthermore, PRL treatment has been shown to increase the number of LH receptors and potentiate the action of LH on the testis (42, 43). However, the testicular LH receptor numbers were decreased in mice lacking GHRs, strongly suggesting that PRL is unable to override the effect of IGF-I deficiency on the number of testicular LH receptors in the absence of IGF-I secretion. Although GH and PRL can up-regulate PRL receptors, high levels of either of these hormones can exert a down-regulation of the PRL receptor expression (44).
Unlike the decreased testosterone response to LH treatment in GHR gene disrupted mice, the basal and LH-stimulated androstenedione secretions were similar to those observed in normal siblings. This suggests that the required biochemical changes responsible for the conversion of androstenedione to testosterone are altered within the testes of GHR-KO mice.
To assess the status of Sertoli cell function, we measured testicular SGP-2 mRNA levels in GHR-KO mice and in their normal siblings. It is known that SGP-2 is secreted by the Sertoli cells of the testis (24, 45). In the present study, this testicular sulfated glycoprotein mRNA levels were not affected by disruption of GHR gene, suggesting that IGF-I may have little role in the production of SGP-2 by the testis. The circulating levels of FSH in GHR-KO mice were significantly decreased and this decreased FSH secretion did not affect SGP-2 mRNA concentrations. A number of studies have shown in rats that FSH treatment had no effects on either SGP-2 secretion or on mRNA expression of this protein (45, 46). Because Sertoli cell secretions influence spermatogenesis (47, 48) and most of male GHR-KO mice are fertile, it is possible that the Sertoli cell function is normal in these mice. It is interesting that the circulating FSH levels in these mice were decreased. This may be possibly due to increased secretion of testicular inhibin, which is known to control FSH secretion (49, 50). Yet, the testicular SGP-2 mRNA levels were not affected by GHR gene disruption suggesting that IGF-I might have a selective stimulatory effect on the secretion of Sertoli cell factors.
In GH-deficient Ames and Snell dwarf mice, the seminiferous tubules are underdeveloped and the spermatogenesis is impaired (51, 52). Furthermore, the plasma FSH levels were reduced in Snell mice (53). Similarly, the circulating FSH levels were significantly reduced in GHR-KO mice. In the present and previous (14) studies, we have reported smaller testes and reduced rate of fertility in these animals. It is known that FSH plays a role in testicular growth and maintenance of spermatogenesis (54). Therefore, it may be reasonable to infer that the growth of testis and possibly spermatogenesis are impaired in GHR gene-disrupted mice due to reduced plasma FSH concentrations and absence of IGF-I secretion. Administration of IGF-I has been shown to increase the number of spermatozoa in the testes of GH-deficient rats (55).
In male mice, it has been demonstrated that experimental induction of hyperprolactinemia results in elevation of circulating LH and FSH levels (40, 43). Despite hyperprolactinemia in GHR-KO mice, the basal plasma LH levels were not altered while FSH secretion was significantly decreased. This altered effect on FSH and LH secretions in these mice suggests that elevated PRL might differentially affect the synthesis and/or release of hypothalamic LHRH and FSHRH that influence LH and FSH secretion respectively. It is tempting to speculate that in the absence of IGF-I secretion, the influence of PRL on pituitary gonadotrope function is altered in GHR-KO mice.
The results of the morphometric analysis indicate that the marked reduction in testicular weight in GHR-KO mice compared with normal mice is primarily due to a very substantial decrease in the length of the seminiferous tubules combined with a decrease in their diameter. Within the seminiferous tubules of GHR-KO mice, the volume of the Sertoli cell nuclei is increased, suggesting a great reduction in the number of germ cells per Sertoli cell. In these mice, the relative volume of Leydig cells (expressed as µg/mg tissue) is not significantly altered, although it is numerically increased by approximately 35%. Because the magnitude of the reduction of the total content (fmol/testis) of LH and PRL receptors (50–62%) exceeds the change in volume density of the Leydig cells (34%), it can be concluded that at least part of the decrease in receptors in GHR-KO mice is not due to the reduction in Leydig cell volume. Importantly, in GHR-KO mice the number of LH and PRL receptors expressed in fmol/mg protein was significantly reduced while the volume of the Leydig cells per mg testis was not significantly altered (but numerically increased). In these GHR gene disrupted mice, the Leydig cell volume per testis was decreased (34%). This together with the significant differences in the percent reduction of LH and PRL receptors further supports this conclusion.
In summary, the present in vivo study clearly demonstrates that LH action on the testis in terms of testosterone secretion is significantly attenuated in GHR gene disrupted mice and suggests that this may be due to a decrease in testicular LH and PRL receptor numbers. These changes are most likely due to the absence of IGF-I in circulation and an increase in PRL secretion. Thus, IGF-I plays a major modulatory effect on testicular endocrine function.
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Acknowledgments |
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Footnotes |
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Abbreviations: GHR, GH receptor; GHR-KO, GHR-knockout; h, human; o, ovine; r, rat; SGP-2, sulfated glycoprotein-2.
Received December 19, 2000.
Accepted for publication April 5, 2001.
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2-macroglobulin, and testibumin in culture media of rat seminiferous
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