1 Geriatric Research, Education and Clincial Center, Veterans Affairs Medical Center and Department of Internal Medicine, Division of Geriatrics, St. Louis University School of Medicine, St. Louis, Missiouri 63106; 2 Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70146; 3 Amgen, Thousand Oaks, California 91320
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ABSTRACT |
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Leptin is a 17-kDa protein, secreted by fat, that controls adiposity and has been proposed to have numerous effects on reproduction in the mouse. To assess whether the effects of leptin on testicular function are direct, we determined whether leptin can cross the murine blood-testis barrier. Multiple time regression analysis showed that a small amount of blood-borne leptin is able to enter the testis but does so by a nonsaturable process. In addition, no significant expression of leptin receptors was found at the Leydig cells or Sertoli cells of the testis. This compares with the presence of a saturable transport system for leptin at the blood-brain barrier and abundant receptors for leptin at the leptomeninges, neurons, and choroid plexus of the central nervous system (CNS). These results support the hypothesis that the effects of leptin on reproductive function are not mediated at the level of the testis but indirectly, probably through the CNS.
OB protein; obesity; reproduction; gonad; transport
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INTRODUCTION |
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LEPTIN is a hormone, secreted by fat cells, (36) that has potent effects on the regulation of body fat and reproductive function (9, 18, 26). Regulation of body fat is believed to be due to the ability of blood-borne leptin to cross the blood-brain barrier (BBB) and to act directly on brain cells such as those in the arcuate nucleus that secrete neuropeptide Y and other satiety factors. A considerable body of evidence supports such regulation, including correlations between levels of leptin in the CSF and serum (10, 28), correlations of CSF and serum levels of leptin with measures of body weight and body fat (10, 14, 28), direct demonstration of a saturable transport system for radioactively labeled leptin across the BBB (4), binding sites for leptin on brain endothelial cells (17) and at the choroid plexus (15, 21), which comprise the vascular and epithelial components of the BBB, and potent effects of leptin on body weight when given directly into the brain even in mice with obesity-induced resistance to peripherally administered leptin (30).
Leptin has been proposed to play a role in murine reproduction. Treatment with leptin restores fertility (11, 23) and ovarian gene expression (35) in the leptin-deficient, obese ob/ob mouse. In normal mice, treatment with leptin leads to the onset of puberty (1) and pregnancy at earlier ages (12), increased levels of follicle-stimulating hormone, increased testicular and seminal vesicle weights, greater seminal vesicle epithelial heights, and elevated sperm counts (5). Leptin alters the release of gonadotrophins from the pituitary (34) and can reverse the starvation-induced changes in the gonadal-pituitary-hypothalamic axis (2). Elevated levels of leptin in the serum during pregnancy (8) are probably due, at least in part, to production of leptin by the placenta (22). However, leptin has not been found to have such widespread effects on reproduction in the rat (13) and hamster (31).
Leptin could exert its putative effects on murine reproduction either directly by acting on the gonads or indirectly by acting at the hypothalamus, at other tissues, or through effects on body weight (7, 33). If leptin acts directly on the testis, then it must be taken up from the blood. However, blood-testis barriers exist (24, 27) that could impede the passage of leptin from the blood into the interstitial space of the testis (vascular barrier) and from the interstitial space into the tubular fluid (Sertoli cell barrier). Substantial, selective uptake of leptin would require saturable transport systems across these barriers similar to those found at the BBB. We investigate here the ability of leptin to be taken up by the testis and whether such uptake is mediated by a distinct transporter.
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MATERIALS AND METHODS |
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Radioactive labeling of leptin and
albumin. Recombinant murine leptin (OB protein) was
obtained from Amgen (Thousand Oaks, CA). The leptin (10 µg) was
radioactively labeled by the Enzymobead method (Bio-Rad, Richmond, CA)
by incubation with 125I overnight
at 4°C. The 125I-leptin
(I-Lep) was separated from unincorporated
125I by purification on a column
of Sephadex G-10. Specific activity was ~102 Ci/g of leptin. The
I-Lep was diluted with an equal volume of lactated Ringer solution with
1% bovine serum albumin (LR-BSA) and was divided into portions and
stored at 70°C until use. Human albumin was labeled with
99mTc (Tc-Alb) with the kit from
Amersham Health Care (San Antonio, TX).
Measurement of influx rates. The rate
of entry of I-Lep into the testis was determined by multiple time
regression analysis (6, 25) and has been previously applied to testis
(3). This method measures the unidirectional influx constant
(Ki, expressed in
ml · g1 · min
1)
and the apparent volume of distribution
(Vi, expressed in µl/g) as
determined by the equation
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(1) |
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(2) |
Adult male ICR mice purchased from Charles River Laboratory
(Wilmington, MA) were used throughout the studies. All studies were
done in a facility approved by the American Association for Accreditation of Laboratory Animal Care under approved animal use
protocols. Mice were allowed to acclimatize with food and water ad libitum for 3 days before studies were begun. Mice were anesthetized with intraperitoneal urethan (40 g/kg), and the jugular vein and carotid artery were exposed. Mice received at
time
0 an intravenous jugular injection of
LR-BSA, the radioactively labeled compounds, and any unlabeled leptin.
Blood obtained from the carotid artery at 1, 2, 5, 7.5, 10, 12.5, 15, or 20 min after the intravenous injection was centrifuged at 5,000 g for 10 min at 4°C, and the level
of radioactivity in the resulting serum was determined in a gamma
counter. The mouse was decapitated immediately after collection of the
blood. The testes were removed and weighed, and their level of
radioactivity was determined in a gamma counter. The brain was also
harvested in some mice, the pituitary and pineal glands were discarded,
and the level of radioactivity was determined in the remainder of the
whole brain.
Characterization of radioactivity entering testis. Acid precipitation was used to determine the portion of the radioactivity in serum and testis that represented I-Lep. It has been shown that acid precipitation gives nearly identical results to those obtained by high-performance liquid chromatography (HPLC) when the percentage of radioactivity that is precipitable is 50% or greater (4). Radioactivity was extracted by homogenizing the whole testis with 1 ml of LR-BSA in a Polytron tissue homogenizer. The homogenate was centrifuged at 5,000 g for 10 min at 4°C. A portion of the resulting supernatant (0.5 ml) was mixed with 0.5 ml of 30% trifluoracetic acid, the mixture was centrifuged at 5,000 g for 10 min at 4°C, and the level of radioactivity in the resulting supernatant (Rs) and precipitate (Rp) was determined. Serum (50 µl) was mixed with 1 ml of LR-BSA and 1 ml of trifluoracetic acid and centrifuged, and the levels of radioactivity in the supernatant and precipitate were determined. The percentage of total counts precipitable (%P) for testis or serum was determined from the equation
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(3) |
In situ hybridization for leptin receptor. In situ hybridization was performed as described by Wilcox (32) on formalin-fixed, paraffin-embedded sections of mouse tissues. Riboprobes were labeled as described in RNase protection assay for leptin receptor except that [33P]rUTP (1,000-3,000 Ci/mol; Amersham) was the labeling isotope. Slides were counterstained with hematoxylin and eosin and photographed with darkfield illumination.
RNase protection assay for leptin receptor. The mouse leptin receptor probes were a generous gift of Eileen Curran (Amgen). The OB receptor (OB-R) common probe (nucleotides 488-780 from Gb:U46135) corresponds to an area of the extracellular domain and detects all forms of the receptor. The OB-Rb (nucleotides 3341-2968 from Gb:U46135) and OB-Ra (nucleotides 2947-3236 from Gb:U42467) are specific for the "b" and "a" forms, respectively. A 105-bp murine cyclophilin probe (Ambion, Austin, TX) was used as an internal control. Radiolabeled antisense transcripts were synthesized from linearized plasmid templates with T7 RNA polymerase (Boehringer-Mannheim, Mannheim, Germany) and [32P]rUTP (800 Ci/mol; Amersham, Arlington Heights, IL). The full-length probes were then purified by electrophoretic separation on a 6% polyacrylamide/7 M urea gel. The hypothalamus and testes were collected from three adult mice, and the tissues were pooled for RNA extraction. The RNA STAT-60 kit (Tel-Test "B", Friendswood, TX) was used to isolate total RNA from adult mouse tissues. The RNAse protection assay was performed with the RPA II kit (Ambion) and 20 µg of total RNA from each sample and 20 µg of yeast total RNA as a negative control. Quantitation was performed with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). The integrated volume of the protected band was normalized to the cyclophilin and charted.
Statistics. Means are presented with their standard errors and were compared by ANOVA followed by the Newman-Keuls multiple comparisons test. Regression lines were calculated by the least squares method and the slope (Ki), intercept (Vi), regression coefficient (r), and level of statistical significance (P) given. Regression lines were compared by Prism (GraphPad Software, San Diego, CA).
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RESULTS |
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Rate of entry into testis. Figure
1 compares the entry rates into testis of
I-Lep and of Tc-Alb. A statistically significant relation occurred
between the testis-to-serum ratio and exposure time for I-Lep
(r = 0.740, n = 23, P < 0.0001), demonstrating that
I-Lep was accumulated by the testis. The
Ki was 0.949 ± 0.193 µl · g1 · min
1,
and the Vi was 8.25 ± 2.86 µl/g. The relation between the testis-to-serum ratio and exposure
time was significant for Tc-Alb (r = 0.607, n = 23, P < 0.01), demonstrating that Tc-Alb
was taken up by whole testis to a measurable degree. For Tc-Alb, the
Ki was 0.260 ± 0.076 µl · g
1 · min
1
and the Vi was 7.17 ± 1.13 µl/g. The regression lines for I-Lep and Tc-Alb were statistically
different [F(1,40) = 11.1, P < 0.005], with the rate of
entry being 3.65 times more for I-Lep than for Tc-Alb.
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Figure 2 shows the serum/testis ratios for
I-Lep corrected for the vascular space as measured by Tc-Alb. The
Ki for entry into
the extravascular space of the testis was 0.689 ± 0.136 µl · g1 · min
1,
and the Vi was 1.07 ± 2.01 µl/g (r = 0.750, n = 23, P < 0.0001).
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Figure 2 also shows the effect of adding 1.0 µg of unlabeled leptin
to the intravenous injection on the rate of entry of I-Lep into testis
after correction for vascular space. Unlabeled leptin had no effect on
I-Lep into the testis. Table 1
compares the regression lines for testis and brain uptake of I-Lep with
and without unlabeled leptin for results corrected for
Tc-Alb.
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Rate of entry into brain. The relation
between brain-to-serum ratios and exposure time was statistically
significant for I-Lep (r = 0.825, n = 22, P < 0.0001) with a
Ki of 0.409 ± 0.063 µl · g1 · min
1
and a Vi of 12.2 ± 0.9 µl/g (Fig. 3). No significant
correlation existed between the brain-to-serum ratios and exposure time
for Tc-Alb (r = 0.167, P > 0.05), demonstrating that Tc-Alb
penetration of the BBB was too low to measure. A statistically
significant difference existed between the I-Lep and Tc-Alb lines:
F(1,40) = 34.8, P < 0.0001. Table 1 shows that 1 µg/mouse of unlabeled leptin inhibited the uptake of I-Lep into brain
for results corrected for Tc-Alb:
[F(1,41) = 10, P < 0.005].
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The percentage of radioactivity that was precipitated by acid (%P) in serum and testis did not vary with time. The mean value when combined for all time points for serum was 83% and for testis was 81%. Acid precipitation of >50% correlates well with the percentage of radioactivity representing intact leptin as determined by HPLC (4).
Passage across vascular and Sertoli cell
barriers. The I-Lep-to-Tc-Alb ratios for serum, TIF,
and SNF are shown in Table 2. ANOVA showed
a statistical difference among the groups:
F(2,12) = 6.73, P < 0.05. The multiple comparisons
test showed that SNF was statistically different from TIF and serum and
that serum and TIF did not differ.
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In situ hybridization. OB-R (common
probe), a probe common to all the splice forms of OB-R, detected a high
level of expression in the brain and localized to the ventral
hypothalamus, choroid plexus, and leptomeninges (Fig.
4, A and
B). In comparison, a moderate level
of expression was detected in small venules in the capsule of the
testis (Fig. 4, C and
D) but not in the seminiferous tubules, interstitial (Leydig) cells, intertubular venules, or capillaries.
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RNase protection assay. The expression
of alternatively spliced leptin receptor transcripts, including the
common form (OB-R, 288 bp) and specific forms (OB-Ra, 289 bp and OB-Rb,
371 bp), is illustrated in Fig. 5, which
shows abundant message in the hypothalamus relative to the testis with
all probes tested. Our findings are in agreement with the low but
detectable mRNA levels of leptin receptor detected in mice by other
authors with various methods, including RNase protection assay (20),
RT-PCR (19, 20), and Northern blotting (16). However, the leptin
receptor expression levels in the testis of mice appear to be lower
than those in the rat (35), which may reflect a species difference.
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DISCUSSION |
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These results show that blood-borne leptin has access to both the brain and to the testis. However, this access is mediated by two different processes. Entry into the brain is by a saturable transport system, whereas for the testis, entry is by leakage.
The testis-blood barrier consists of a vascular barrier, separating the circulation from the testicular interstitial space, and the less permeable Sertoli cell barrier, separating the interstitial space from the seminiferous tubule fluid (24, 27). Some substances are unable to cross either of these barriers, other substances can only cross the vascular barrier, and other substances can cross both barriers. The vascular barrier of the testis restricts the entry of serum proteins such as albumin but not to the extent that the vascular barrier of the brain does. Our results are consistent with that observation, as we could measure the uptake of Tc-Alb by the testis but not by the brain.
In comparison with Tc-Alb, I-Lep was taken up by both the testis and brain. The entry rate of I-Lep into the testis was 3.65 times greater than the entry rate of Tc-Alb. Because leptin is about one-fourth the size of Tc-Alb, this difference could be due to the ability of the smaller molecule to leak more easily into the testis. This compares with the uptake of I-Lep by the brain, which is 20 times greater than the uptake by the brain of Tc-Alb. That the uptake of I-Lep into the testis is due to leakage rather than to a saturable transport was confirmed here by showing that a dose of unlabeled leptin capable of inhibiting brain uptake had no effect on the uptake by testis.
The uptake of I-Lep by the testis can also be compared with that of
interleukin-1 (IL-1), a cytokine of similar molecular weight as
leptin. The uptake of IL-1 from the blood into the testis is due, in
part to a saturable transport (3). The
Ki for
radioactively labeled IL-1 (1.81 µl/g-min) is about twice the rate of
that found here for I-Lep.
Radioactivity recovered from testis could be precipitated with acid, confirming that it represented intact I-Lep. Previous results have shown that for I-Lep, acid precipitation and HPLC give nearly identical results (4).
Table 2 demonstrates a progressive partitioning of I-Lep relative to Tc-Alb with passage across the vascular barrier from serum to TIF and across the Sertoli cell barrier from TIF to SNF. This results in about a fourfold increase in the I-Lep-to-Tc-Alb ratio in SNF compared with that of serum. It is unclear whether this concentration of I-Lep in SNF is due to leakage or saturable transport at the Sertoli cell barrier.
Consistent with the lack of demonstration by pharmacokinetic methods of any saturable transport of leptin into the testis was the lack of demonstration of leptin receptor mRNA in the testicular parenchyma, including the intratesticular vascular structures, seminiferous tubules, or interstitial (Leydig) cells by in situ hybridization (Fig. 4D). A small amount of leptin receptor mRNA was located at the venules of the capsule. The small amount of leptin receptor mRNA detected in the mouse testis by the RNase protection assay (Fig. 5) in this study is entirely consistent with the mRNA levels detected in mice by others with various methods, including RNase protection assay (20), RT-PCR (19, 20), and Northern blotting (16). Moreover, the localization of the leptin receptor signal to the capsular vessels by in situ hybridization suggests that the vascular structures may be a major source of leptin receptor message in testicular tissue homogenates from mice. However, the leptin receptor expression levels in the testis of mice appear to be lower (by RT-PCR) than those in the rat (35), which may reflect a species difference. Without receptors, leptin could not induce intracellular signaling or be transported at the intraductal vasculature, Leydig cells, or Sertoli cells. By contrast, we did find significant receptor expression within the CNS by both methods (Fig. 4B; Fig. 5).
The lack of a saturable transport mechanism across the blood-testis barrier and the inability to demonstrate leptin receptors within the testis are consistent with the hypothesis that leptin does not act directly at the testes to induce its effects on reproduction.
In conclusion, these results show that although blood-borne leptin has access to the testis and its various fluid compartments, the major mechanism for uptake of leptin by the testis is leakage. The lack of a saturable transport system and leptin receptors within the testis is consistent with leptin mediating its effects on reproduction by indirect mechanisms.
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ACKNOWLEDGEMENTS |
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This study was supported by the Veterans Affairs Merit Review.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. A. Banks, 915 N. Grand Blvd, St. Louis, MO 63106 (E-mail: bankswa{at}slu.edu).
Received 6 July 1998; accepted in final form 19 February 1999.
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