Importance of the Paraventricular Nucleus of the Hypothalamus as a Component of a Neural Pathway between the Brain and the Testes that Modulates Testo
Abstractazo, http://www.100md.com
We previously reported that in adult male rats, the intracerebroventricular (icv) injection of corticotropin-releasing factor (CRF) or the ß-adrenergic agonist isoproterenol (ISO) significantly inhibited the ability of human chorionic gonadotropin (hCG) to stimulate testosterone (T) secretion. The finding that this phenomenon also took place when LH release had been blocked with an LHRH antagonist suggested that icv CRF and ISO did not alter Leydig cell function by influencing the activity of pituitary gonadotrophs. We therefore proposed the existence of a neural pathway connecting the brain to the testes, whose activation by icv CRF or ISO interfered with T secretion. Based on the intratesticular injection of the transganglionic tracer pseudorabies virus, we recently identified the paraventricular nucleus (PVN) of the hypothalamus as a component of this neural link. The aim of the present work was to investigate the functional role of this brain area in mediating the ability of CRF and ISO to inhibit the ability of hCG to stimulate T secretion. We first demonstrated that local microinfusion of CRF or ISO directly into the PVN mimicked the effect of their icv injection, suggesting that the PVN does indeed represent a site of action of ISO and CRF in altering Leydig cell responsiveness to gonadotropin. In contrast, neither CRF nor ISO microinfusion into the central amygdala or the frontal cortex influenced hCG-stimulated T secretion. To further investigate the role of the PVN in ISO- and CRF-induced blunting of hCG stimulation of T, we determined the effect of icv CRF or ISO on testicular activity of rats with electrolytic lesions of the PVN. These lesions, which did not in themselves influence Leydig cell responsiveness to hCG, blocked the effect of both icv ISO and CRF on hCG-induced T release. Collectively, these results support the hypothesis that CRF- and ISO-induced activation of cells in the area of the PVN decreases the ability of gonadotropin to release T and suggests that this nucleus represents an important site of the proposed neural connection between the brain and the testes.
Introductionll5\m, 百拇医药
IN ADDITION to being regulated by the well studied LHRH-LH pathway, testosterone (T) synthesis and secretion are influenced by a number of less well understood mechanisms (1, 2, 3). These pathways, which are often responsive to stressful stimuli, regulate T release through hormones and neurosecretagogues that are present in the circulation and/or synthesized and secreted within the testis and directly influence steroidogenic enzymes (1, 2, 3, 4, 5, 6). Although the ability of some acute stressors to lower plasma T levels appears to involve at least in part decreased activity of the LHRH-LH system (3, 7, 8), it has long been known that other noxious stimuli interfere with T release in the absence of measurable changes in mean LH levels (4, 5, 6). This has been interpreted as evidence for the existence of other control mechanisms that regulate Leydig cell function independently of the pituitary (9, 10, 11, 12). We recently showed the existence of a neurally based regulatory pathway that bypasses the LH-LHRH system to influence T production and is dependent on ß-adrenergic receptor activation in the brain (13, 14). Specifically, we found that the intracerebroventricular (icv) injection of compounds known to increase hypothalamic catecholamine concentration, such as corticotropin-releasing factor (CRF) and IL-1ß, blunted human chorionic gonadotropin (hCG)-induced T secretion by decreasing testicular levels of the steroidogenic acute regulatory protein (15), which plays a critical role for T synthesis in the testes (16). This effect did not seem to be due to sympathetically mediated increased vasoconstriction and decreased blood flow to the testes, as histological examination of the testes after icv injection of either drug did not indicate decreased vascularization, changes in testicular enzyme activity were not general but restricted to the steroidogenic acute regulatory protein and not other steroidogenic enzymes, and reductions in blood flow to the testis by testicular nerve stimulation are under the control of {alpha} -, not ß-adrenergic pathways (17).
We (18) and others (19) recently provided anatomical evidence for the proposed inhibitory neural pathway between the brain and the testis by showing that the intratesticular injection of the transneuronal tracer pseudorabies virus (PRV), which is retrogradely transported from neurons innervating the end organ into which it is administered (20), resulted in labeled cells in the paraventricular nucleus of the hypothalamus (PVN), the central amygdala (CeAM), and the A5 noradrenergic group of the brainstem, among others. The purpose of the present work was to further elucidate the neural sites involved in the rapid inhibition of T secretion by icv injected isoproterenol (ISO) and CRF. We microinfused these reagents into brain areas that PRV studies had identified as possible components of the brain-testes neural pathway, namely, the PVN, the CeAM, and, as a control, the frontal cortex. Because microinfusion of ISO or CRF into the PVN was found to blunt hCG-induced T secretion in the first set of studies, we then examined the ability of electrolytic lesions of the PVN to prevent the influence of icv administered ISO or CRF on hCG-induced stimulation of T secretion. The results of these experiments point to a crucial role for the PVN in the rapid inhibition of Leydig cell responsiveness to gonadotropin by ISO and CRF and, by extension, as a major part of the pathway by which the brain directly regulates testicular function via a neural connection independently of the pituitary.
Materials and Methodsjcx, 百拇医药
Animalsjcx, 百拇医药
Adult male rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), weighing 180–200 g on arrival, were housed individually after any surgical treatment under controlled lighting conditions (12 h of light, 12 h of darkness) with food and water available ad libitum. All protocols were approved by the institutional animal care and use committee of the Salk Institute.jcx, 百拇医药
Microinfusion of ISO or CRFjcx, 百拇医药
Deeply anesthetized animals were placed in a stereotaxic surgery apparatus, and bilateral guide cannulas were inserted directly above the PVN, amygdala, or frontal cortex. The coordinates used from bregma were: for the PVN: anterio-posterior, -1.8 mm; lateral, ±0.5 mm; dorso-ventral, -7.4 mm; for the amygdala: anterio-posterior, -2.1 mm; lateral, ±3.8 mm; dorso-ventral, -7.4 mm; and for the frontal cortex: anterio-posterior, +3.2 mm; lateral, -0.8 mm; dorso-ventral, -4.2 mm. The cannulas were held in place by small machine screws and plastic dental cement. They were kept patent by insertion of indwelling stylets. After a 7- to 10-d recovery time, the animals were fitted with a jugular venous iv cannulas and allowed to recover an additional 2–3 d. On the day of experimentation, 1 µl ISO (1 µg/side, in a 1-µl total volume; purchased from Sigma-Aldrich, St. Louis, MO), rat/human CRF (0.5 µg/side, in a 1-µl total volume) (21), or vehicle (apyrogenic saline) was infused into the PVN, CeAM, or frontal cortex over 5 min using an automated microinfusion pump (Harvard Instruments, Cambridge, MA). Several doses of ISO and CRF were tested in preliminary studies, and we chose those that provided consistent inhibition of Leydig cell function. Twenty minutes after ISO, CRF, or vehicle microinfusion, hCG (1 IU/kg; purchased from Sigma-Aldrich) was injected iv. Blood samples (0.3 ml) were taken before microinfusion of ISO or CRF to obtain basal T secretion values as well as 20, 45, and 90 min after iv hCG injection.
PVN lesions/':, http://www.100md.com
Bilateral electrolytic lesions of the PVN were made by passing a 0.8-mamp current for 35 sec through a tungsten-tipped electrode (A-M Systems, Inc., Carlsborg, WA). The coordinates for electrode placement from bregma were: anterio-posterior, -1.65 mm; lateral, ±0.4 mm; and dorso-ventral, -8.0 mm. Sham lesions were performed using the same coordinates whereby the electrode was lowered -5 mm dorso-ventral, and no current passed was through it. All animals were also fitted with icv cannula (coordinates: anterio-posterior, -0.4 mm; lateral, ±1.4 mm; DV, -3.8 mm) and, 7–8 d later, an iv cannula. On the day of experimentation, ISO (4.8 µg), CRF (2 µg), or the vehicle was infused icv in a total volume of 5 µl. Twenty minutes later, hCG (1 IU/kg) was injected iv. Blood samples were drawn before icv injection, and 20, 45, and 90 min after hCG injection./':, http://www.100md.com
Experimental protocol/':, http://www.100md.com
On the day of experimentation, animals were removed to a soundproof room and housed individually in opaque buckets with their cannula and catheters connected by polyethylene tubing to syringes so that treatments could be administered without disturbing the animals. All injections were giving at least 2 h after rehousing to allow hormone levels to return to normal. After experimentation, animals were transcardially perfused with 0.4% paraformaldehyde (PFA). Brains were removed and stored in PFA until overnight cryoprotection in a solution of 10% sucrose in 4% PFA, then cut at 30 µm on a cryostat. Placements of cannula and PVN lesions were checked after Nissl staining of sections. Only animals with appropriately placed cannula/lesions were used in the statistical analysis of the data.
T measurement\;2, http://www.100md.com
T was measured in 50-µl duplicate unextracted plasma samples using a commercially available, solid phase RIA kit (Diagnostic Products, Los Angeles, CA). The characteristics of this assay have been previously described (14).\;2, http://www.100md.com
Statistical analysis\;2, http://www.100md.com
Data were analyzed by one-way ANOVA with repeated measures and by t or Bonferroni/Dunn test.\;2, http://www.100md.com
Results\;2, http://www.100md.com
Microinfusion of ISO or CRF into the PVN, CeAM, and frontal cortex\;2, http://www.100md.com
The microinfusion of ISO (1 µg/side) or CRF (0.5 µg/side) directly into the PVN significantly attenuated Leydig cell responsiveness to hCG-induced T secretion compared with vehicle-treated rats (Figs. 1 and 2). In contrast, the microinfusion of the same amounts of ISO or CRF directly into the frontal cortex or CeAM did not attenuate hCG-induced T secretion (Figs. 3–5).\;2, http://www.100md.com
fig.ommitteed
Figure 1. Microinfusion of ISO (1 µg/side) directly into the PVN blunted hCG-induced T secretion. ISO was microinfused into the PVN 20 min before iv hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. In Figs. 1–7, the left panel illustrates T release as a function of time after hCG injection, whereas the right panel illustrates cumulative T levels measured at the 20, 45, and 90 min points. Each point or bar represents the mean ± SEM of six to eight animals. **, P < 0.01 from vehicle/hCG.0m)70, 百拇医药
fig.ommitteed0m)70, 百拇医药
Figure 2. Microinfusion of CRF (0.5 µg/side) directly into the PVN blunted hCG-induced T secretion. CRF was microinfused into the PVN 20 min before iv hCG (1 U/kg) administration. Blood for basal T measurement was taken before CRF microinfusion. Each point or bar represents the mean ± SEM of six to eight animals. *, P < 0.05; **, P < 0.01 (vs. vehicle/hCG).0m)70, 百拇医药
fig.ommitteed0m)70, 百拇医药
Figure 3. Microinfusion of ISO (1 µg/side) directly into the CeAM did not have an effect on hCG-induced T secretion. ISO was microinfused into the CeAM 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. Each point or bar represents the mean ± SEM of four to six animals. **, P < 0.01 (vs. vehicle/hCG).
fig.ommitteed!y%nf, 百拇医药
Figure 4. Microinfusion of ISO (1 µg/side) into the frontal cortex did not affect hCG-induced T secretion. ISO was microinfused into the frontal cortex 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. Each point or bar represents the mean ± SEM of four animals. *, P < 0.05; **, P < 0.01 (vs. vehicle/hCG).!y%nf, 百拇医药
fig.ommitteed!y%nf, 百拇医药
Figure 5. Microinfusion of CRF (0.5 µg/side) into the CeAM or frontal cortex did not affect hCG-induced T secretion. CRF was microinfused into the frontal cortex 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before CRF microinfusion. Each point or bar represents the mean ± SEM of six to eight animals.!y%nf, 百拇医药
PVN lesions!y%nf, 百拇医药
By themselves, PVN lesions did not alter Leydig cell responsiveness to hCG, but blocked the ability of icv ISO (4.8 µg) or CRF (2 µg) to attenuate the ability of hCG to stimulate T secretion (Figs. 6 and 7). In contrast, sham lesions did not affect Leydig cell responsiveness to gonadotropin or the ability of icv ISO to blunt this effect.
fig.ommitteedcv, 百拇医药
Figure 6. PVN lesions (PVN-X) did not affect hCG-induced T synthesis and secretion, but blocked the effect of icv ISO on hCG-induced T secretion. Blood for basal T measurement was drawn before the icv administration of ISO (4.8 µG). hCG (1 U/kg) was administered 20 min after ISO. Each point or bar represents the mean ± SEM of seven or eight animals. *, P < 0.05; **, P < 0.01.cv, 百拇医药
fig.ommitteedcv, 百拇医药
Figure 7. PVN lesions (PVN-X) did not affect hCG-induced T synthesis and secretion, but blocked the effect of icv CRF on hCG-induced T secretion. Blood for basal T measurement was drawn before the icv administration of CRF (2 µg). hCG (1 U/kg) was administered 20 min after CRF. Each point or bar represents the mean ± SEM of six to eight animals. **, P < 0.01.cv, 百拇医药
Histologycv, 百拇医药
Figure 8 illustrates a composite schematic of PVN lesions that were effective in blocking the ability of icv ISO to blunt hCG stimulation of T.cv, 百拇医药
fig.ommitteed
Figure 8. Schematic showing the areas of PVN lesions that were effective in blocking the inhibitory effect of icv ISO or CRF on hCG-induced T secretion. Each set of three schematics represents the rostral-caudal extent of a single lesion through the PVN.}f6|, 百拇医药
Discussion}f6|, 百拇医药
In the present studies we show that microinfusion of the ß-adrenergic agonist ISO or of CRF directly into the PVN blunted Leydig cell responsiveness to gonadotropin. In contrast, ISO or CRF microinfusion into the CeAM or frontal cortex had no effect on the stimulation of T secretion by hCG. These results confirm our previous findings that delivery of ISO and CRF into the brain inhibits Leydig cell activity, a response that is dissociated from any peripheral influence that these secretagogues might have when they are delivered to the gonads via the bloodstream and that is distinct from their influence on LH release (13, 14). Because the influence of icv injected ISO or CRF could be reversed by central blockade of ß-adrenergic receptors (18), we had proposed that activation of bioamine-dependent pathways, for example by stress, provided a novel mechanism controlling T secretion that was independent of the LHRH-LH axis.
On the basis of PRV-based transganglionic techniques, we (18) and others (19) recently reported that the PVN represented a critical region of a neural hypothalamic-testicular circuit. This concept received support not only from the ability of ISO or CRF, microinfused into this region, to interfere with Leydig cell activity, but also from our present finding that PVN lesions abolished the influence of icv injected compounds. Until now, the PVN has not been regarded as an important part of the pathways that regulate reproductive functions. For example, we had shown that destruction of the PVN did not modify the influence of stress on LH secretion (17), which agrees with the fact that most LHRH neurons are found in the medial preoptic area (8). The data illustrated here therefore provide novel information regarding an unexpected role of the PVN in contributing to the neurocircuitry controlling T secretion. One possibility pertains to the fact that cells originating in the PVN send projections to the A5 noradrenergic cell group of the medulla, which also contains neurons transynaptically linked to the testis (10, 22). This area may therefore be part of the brain-testicular neurocircuitry that is activated by icv injected CRF and ISO, a hypothesis that is presently under investigation in our laboratory.
In conclusion, the data from our previous studies (13, 14) combined with those reported here form a strong case for the existence of an inhibitory, catecholamine-activated, steroidogenic regulatory pathway that is independent of the pituitary and in which the PVN plays a major role. These results should increase our understanding of the mechanisms through which stress and immune system activation, which increase CRF and catecholamine levels in the PVN (23, 24, 25), influence testicular function.4, 百拇医药
Acknowledgments4, 百拇医药
The authors are indebted to Dr. Jean Rivier (The Salk Institute, La Jolla, CA) for the gift of synthetic rat/human CRF.4, 百拇医药
Received July 30, 2002.4, 百拇医药
Accepted for publication October 31, 2002.4, 百拇医药
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Ogilvie K, Hales K, Roberts M, Hales D, Rivier C 1999 The inhibitory effect of intracerebroventricularly injected interleukin 1ß on testosterone secretion in the rat: role of steroidogenic acute regulatory protein. Biol Reprod 60:527–533'5, 百拇医药
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We previously reported that in adult male rats, the intracerebroventricular (icv) injection of corticotropin-releasing factor (CRF) or the ß-adrenergic agonist isoproterenol (ISO) significantly inhibited the ability of human chorionic gonadotropin (hCG) to stimulate testosterone (T) secretion. The finding that this phenomenon also took place when LH release had been blocked with an LHRH antagonist suggested that icv CRF and ISO did not alter Leydig cell function by influencing the activity of pituitary gonadotrophs. We therefore proposed the existence of a neural pathway connecting the brain to the testes, whose activation by icv CRF or ISO interfered with T secretion. Based on the intratesticular injection of the transganglionic tracer pseudorabies virus, we recently identified the paraventricular nucleus (PVN) of the hypothalamus as a component of this neural link. The aim of the present work was to investigate the functional role of this brain area in mediating the ability of CRF and ISO to inhibit the ability of hCG to stimulate T secretion. We first demonstrated that local microinfusion of CRF or ISO directly into the PVN mimicked the effect of their icv injection, suggesting that the PVN does indeed represent a site of action of ISO and CRF in altering Leydig cell responsiveness to gonadotropin. In contrast, neither CRF nor ISO microinfusion into the central amygdala or the frontal cortex influenced hCG-stimulated T secretion. To further investigate the role of the PVN in ISO- and CRF-induced blunting of hCG stimulation of T, we determined the effect of icv CRF or ISO on testicular activity of rats with electrolytic lesions of the PVN. These lesions, which did not in themselves influence Leydig cell responsiveness to hCG, blocked the effect of both icv ISO and CRF on hCG-induced T release. Collectively, these results support the hypothesis that CRF- and ISO-induced activation of cells in the area of the PVN decreases the ability of gonadotropin to release T and suggests that this nucleus represents an important site of the proposed neural connection between the brain and the testes.
Introductionll5\m, 百拇医药
IN ADDITION to being regulated by the well studied LHRH-LH pathway, testosterone (T) synthesis and secretion are influenced by a number of less well understood mechanisms (1, 2, 3). These pathways, which are often responsive to stressful stimuli, regulate T release through hormones and neurosecretagogues that are present in the circulation and/or synthesized and secreted within the testis and directly influence steroidogenic enzymes (1, 2, 3, 4, 5, 6). Although the ability of some acute stressors to lower plasma T levels appears to involve at least in part decreased activity of the LHRH-LH system (3, 7, 8), it has long been known that other noxious stimuli interfere with T release in the absence of measurable changes in mean LH levels (4, 5, 6). This has been interpreted as evidence for the existence of other control mechanisms that regulate Leydig cell function independently of the pituitary (9, 10, 11, 12). We recently showed the existence of a neurally based regulatory pathway that bypasses the LH-LHRH system to influence T production and is dependent on ß-adrenergic receptor activation in the brain (13, 14). Specifically, we found that the intracerebroventricular (icv) injection of compounds known to increase hypothalamic catecholamine concentration, such as corticotropin-releasing factor (CRF) and IL-1ß, blunted human chorionic gonadotropin (hCG)-induced T secretion by decreasing testicular levels of the steroidogenic acute regulatory protein (15), which plays a critical role for T synthesis in the testes (16). This effect did not seem to be due to sympathetically mediated increased vasoconstriction and decreased blood flow to the testes, as histological examination of the testes after icv injection of either drug did not indicate decreased vascularization, changes in testicular enzyme activity were not general but restricted to the steroidogenic acute regulatory protein and not other steroidogenic enzymes, and reductions in blood flow to the testis by testicular nerve stimulation are under the control of {alpha} -, not ß-adrenergic pathways (17).
We (18) and others (19) recently provided anatomical evidence for the proposed inhibitory neural pathway between the brain and the testis by showing that the intratesticular injection of the transneuronal tracer pseudorabies virus (PRV), which is retrogradely transported from neurons innervating the end organ into which it is administered (20), resulted in labeled cells in the paraventricular nucleus of the hypothalamus (PVN), the central amygdala (CeAM), and the A5 noradrenergic group of the brainstem, among others. The purpose of the present work was to further elucidate the neural sites involved in the rapid inhibition of T secretion by icv injected isoproterenol (ISO) and CRF. We microinfused these reagents into brain areas that PRV studies had identified as possible components of the brain-testes neural pathway, namely, the PVN, the CeAM, and, as a control, the frontal cortex. Because microinfusion of ISO or CRF into the PVN was found to blunt hCG-induced T secretion in the first set of studies, we then examined the ability of electrolytic lesions of the PVN to prevent the influence of icv administered ISO or CRF on hCG-induced stimulation of T secretion. The results of these experiments point to a crucial role for the PVN in the rapid inhibition of Leydig cell responsiveness to gonadotropin by ISO and CRF and, by extension, as a major part of the pathway by which the brain directly regulates testicular function via a neural connection independently of the pituitary.
Materials and Methodsjcx, 百拇医药
Animalsjcx, 百拇医药
Adult male rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), weighing 180–200 g on arrival, were housed individually after any surgical treatment under controlled lighting conditions (12 h of light, 12 h of darkness) with food and water available ad libitum. All protocols were approved by the institutional animal care and use committee of the Salk Institute.jcx, 百拇医药
Microinfusion of ISO or CRFjcx, 百拇医药
Deeply anesthetized animals were placed in a stereotaxic surgery apparatus, and bilateral guide cannulas were inserted directly above the PVN, amygdala, or frontal cortex. The coordinates used from bregma were: for the PVN: anterio-posterior, -1.8 mm; lateral, ±0.5 mm; dorso-ventral, -7.4 mm; for the amygdala: anterio-posterior, -2.1 mm; lateral, ±3.8 mm; dorso-ventral, -7.4 mm; and for the frontal cortex: anterio-posterior, +3.2 mm; lateral, -0.8 mm; dorso-ventral, -4.2 mm. The cannulas were held in place by small machine screws and plastic dental cement. They were kept patent by insertion of indwelling stylets. After a 7- to 10-d recovery time, the animals were fitted with a jugular venous iv cannulas and allowed to recover an additional 2–3 d. On the day of experimentation, 1 µl ISO (1 µg/side, in a 1-µl total volume; purchased from Sigma-Aldrich, St. Louis, MO), rat/human CRF (0.5 µg/side, in a 1-µl total volume) (21), or vehicle (apyrogenic saline) was infused into the PVN, CeAM, or frontal cortex over 5 min using an automated microinfusion pump (Harvard Instruments, Cambridge, MA). Several doses of ISO and CRF were tested in preliminary studies, and we chose those that provided consistent inhibition of Leydig cell function. Twenty minutes after ISO, CRF, or vehicle microinfusion, hCG (1 IU/kg; purchased from Sigma-Aldrich) was injected iv. Blood samples (0.3 ml) were taken before microinfusion of ISO or CRF to obtain basal T secretion values as well as 20, 45, and 90 min after iv hCG injection.
PVN lesions/':, http://www.100md.com
Bilateral electrolytic lesions of the PVN were made by passing a 0.8-mamp current for 35 sec through a tungsten-tipped electrode (A-M Systems, Inc., Carlsborg, WA). The coordinates for electrode placement from bregma were: anterio-posterior, -1.65 mm; lateral, ±0.4 mm; and dorso-ventral, -8.0 mm. Sham lesions were performed using the same coordinates whereby the electrode was lowered -5 mm dorso-ventral, and no current passed was through it. All animals were also fitted with icv cannula (coordinates: anterio-posterior, -0.4 mm; lateral, ±1.4 mm; DV, -3.8 mm) and, 7–8 d later, an iv cannula. On the day of experimentation, ISO (4.8 µg), CRF (2 µg), or the vehicle was infused icv in a total volume of 5 µl. Twenty minutes later, hCG (1 IU/kg) was injected iv. Blood samples were drawn before icv injection, and 20, 45, and 90 min after hCG injection./':, http://www.100md.com
Experimental protocol/':, http://www.100md.com
On the day of experimentation, animals were removed to a soundproof room and housed individually in opaque buckets with their cannula and catheters connected by polyethylene tubing to syringes so that treatments could be administered without disturbing the animals. All injections were giving at least 2 h after rehousing to allow hormone levels to return to normal. After experimentation, animals were transcardially perfused with 0.4% paraformaldehyde (PFA). Brains were removed and stored in PFA until overnight cryoprotection in a solution of 10% sucrose in 4% PFA, then cut at 30 µm on a cryostat. Placements of cannula and PVN lesions were checked after Nissl staining of sections. Only animals with appropriately placed cannula/lesions were used in the statistical analysis of the data.
T measurement\;2, http://www.100md.com
T was measured in 50-µl duplicate unextracted plasma samples using a commercially available, solid phase RIA kit (Diagnostic Products, Los Angeles, CA). The characteristics of this assay have been previously described (14).\;2, http://www.100md.com
Statistical analysis\;2, http://www.100md.com
Data were analyzed by one-way ANOVA with repeated measures and by t or Bonferroni/Dunn test.\;2, http://www.100md.com
Results\;2, http://www.100md.com
Microinfusion of ISO or CRF into the PVN, CeAM, and frontal cortex\;2, http://www.100md.com
The microinfusion of ISO (1 µg/side) or CRF (0.5 µg/side) directly into the PVN significantly attenuated Leydig cell responsiveness to hCG-induced T secretion compared with vehicle-treated rats (Figs. 1 and 2). In contrast, the microinfusion of the same amounts of ISO or CRF directly into the frontal cortex or CeAM did not attenuate hCG-induced T secretion (Figs. 3–5).\;2, http://www.100md.com
fig.ommitteed
Figure 1. Microinfusion of ISO (1 µg/side) directly into the PVN blunted hCG-induced T secretion. ISO was microinfused into the PVN 20 min before iv hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. In Figs. 1–7, the left panel illustrates T release as a function of time after hCG injection, whereas the right panel illustrates cumulative T levels measured at the 20, 45, and 90 min points. Each point or bar represents the mean ± SEM of six to eight animals. **, P < 0.01 from vehicle/hCG.0m)70, 百拇医药
fig.ommitteed0m)70, 百拇医药
Figure 2. Microinfusion of CRF (0.5 µg/side) directly into the PVN blunted hCG-induced T secretion. CRF was microinfused into the PVN 20 min before iv hCG (1 U/kg) administration. Blood for basal T measurement was taken before CRF microinfusion. Each point or bar represents the mean ± SEM of six to eight animals. *, P < 0.05; **, P < 0.01 (vs. vehicle/hCG).0m)70, 百拇医药
fig.ommitteed0m)70, 百拇医药
Figure 3. Microinfusion of ISO (1 µg/side) directly into the CeAM did not have an effect on hCG-induced T secretion. ISO was microinfused into the CeAM 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. Each point or bar represents the mean ± SEM of four to six animals. **, P < 0.01 (vs. vehicle/hCG).
fig.ommitteed!y%nf, 百拇医药
Figure 4. Microinfusion of ISO (1 µg/side) into the frontal cortex did not affect hCG-induced T secretion. ISO was microinfused into the frontal cortex 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before ISO microinfusion. Each point or bar represents the mean ± SEM of four animals. *, P < 0.05; **, P < 0.01 (vs. vehicle/hCG).!y%nf, 百拇医药
fig.ommitteed!y%nf, 百拇医药
Figure 5. Microinfusion of CRF (0.5 µg/side) into the CeAM or frontal cortex did not affect hCG-induced T secretion. CRF was microinfused into the frontal cortex 20 min before hCG (1 U/kg) administration. Blood for basal T measurement was taken before CRF microinfusion. Each point or bar represents the mean ± SEM of six to eight animals.!y%nf, 百拇医药
PVN lesions!y%nf, 百拇医药
By themselves, PVN lesions did not alter Leydig cell responsiveness to hCG, but blocked the ability of icv ISO (4.8 µg) or CRF (2 µg) to attenuate the ability of hCG to stimulate T secretion (Figs. 6 and 7). In contrast, sham lesions did not affect Leydig cell responsiveness to gonadotropin or the ability of icv ISO to blunt this effect.
fig.ommitteedcv, 百拇医药
Figure 6. PVN lesions (PVN-X) did not affect hCG-induced T synthesis and secretion, but blocked the effect of icv ISO on hCG-induced T secretion. Blood for basal T measurement was drawn before the icv administration of ISO (4.8 µG). hCG (1 U/kg) was administered 20 min after ISO. Each point or bar represents the mean ± SEM of seven or eight animals. *, P < 0.05; **, P < 0.01.cv, 百拇医药
fig.ommitteedcv, 百拇医药
Figure 7. PVN lesions (PVN-X) did not affect hCG-induced T synthesis and secretion, but blocked the effect of icv CRF on hCG-induced T secretion. Blood for basal T measurement was drawn before the icv administration of CRF (2 µg). hCG (1 U/kg) was administered 20 min after CRF. Each point or bar represents the mean ± SEM of six to eight animals. **, P < 0.01.cv, 百拇医药
Histologycv, 百拇医药
Figure 8 illustrates a composite schematic of PVN lesions that were effective in blocking the ability of icv ISO to blunt hCG stimulation of T.cv, 百拇医药
fig.ommitteed
Figure 8. Schematic showing the areas of PVN lesions that were effective in blocking the inhibitory effect of icv ISO or CRF on hCG-induced T secretion. Each set of three schematics represents the rostral-caudal extent of a single lesion through the PVN.}f6|, 百拇医药
Discussion}f6|, 百拇医药
In the present studies we show that microinfusion of the ß-adrenergic agonist ISO or of CRF directly into the PVN blunted Leydig cell responsiveness to gonadotropin. In contrast, ISO or CRF microinfusion into the CeAM or frontal cortex had no effect on the stimulation of T secretion by hCG. These results confirm our previous findings that delivery of ISO and CRF into the brain inhibits Leydig cell activity, a response that is dissociated from any peripheral influence that these secretagogues might have when they are delivered to the gonads via the bloodstream and that is distinct from their influence on LH release (13, 14). Because the influence of icv injected ISO or CRF could be reversed by central blockade of ß-adrenergic receptors (18), we had proposed that activation of bioamine-dependent pathways, for example by stress, provided a novel mechanism controlling T secretion that was independent of the LHRH-LH axis.
On the basis of PRV-based transganglionic techniques, we (18) and others (19) recently reported that the PVN represented a critical region of a neural hypothalamic-testicular circuit. This concept received support not only from the ability of ISO or CRF, microinfused into this region, to interfere with Leydig cell activity, but also from our present finding that PVN lesions abolished the influence of icv injected compounds. Until now, the PVN has not been regarded as an important part of the pathways that regulate reproductive functions. For example, we had shown that destruction of the PVN did not modify the influence of stress on LH secretion (17), which agrees with the fact that most LHRH neurons are found in the medial preoptic area (8). The data illustrated here therefore provide novel information regarding an unexpected role of the PVN in contributing to the neurocircuitry controlling T secretion. One possibility pertains to the fact that cells originating in the PVN send projections to the A5 noradrenergic cell group of the medulla, which also contains neurons transynaptically linked to the testis (10, 22). This area may therefore be part of the brain-testicular neurocircuitry that is activated by icv injected CRF and ISO, a hypothesis that is presently under investigation in our laboratory.
In conclusion, the data from our previous studies (13, 14) combined with those reported here form a strong case for the existence of an inhibitory, catecholamine-activated, steroidogenic regulatory pathway that is independent of the pituitary and in which the PVN plays a major role. These results should increase our understanding of the mechanisms through which stress and immune system activation, which increase CRF and catecholamine levels in the PVN (23, 24, 25), influence testicular function.4, 百拇医药
Acknowledgments4, 百拇医药
The authors are indebted to Dr. Jean Rivier (The Salk Institute, La Jolla, CA) for the gift of synthetic rat/human CRF.4, 百拇医药
Received July 30, 2002.4, 百拇医药
Accepted for publication October 31, 2002.4, 百拇医药
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