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Departments of Physiology (D.F.B., F.J.K.) and Biology (L.A.T.), Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Dr. Fred J. Karsch, Reproductive Sciences Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan 48109-0404. E-mail: fjkarsch{at}umich.edu
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Introduction |
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Using a rodent model, one study has shown that interleukin-1 (IL-1), a cytokine important in mediating responses to immune challenge, stimulates CRH secretion in portal blood, as assessed in one sample collected over 30 min, but does not significantly alter AVP (7). Other studies in rodents provide evidence that this same cytokine, or CRH, delivered centrally can inhibit GnRH release (5, 8). Importantly, these earlier in vivo studies directly assessing neuropeptide secretion involved anesthetized animals and terminal surgical intervention, manipulations that could profoundly impact the neuroendocrine stress and reproductive axes in and of themselves. More recent studies in rodents and Shiba goats, which circumvent these technical difficulties, suggest that immune challenge inhibits hypothalamic multiunit activity associated with LH pulsatile suppression, although neither GnRH nor the stress neuropeptides were monitored (9, 10). Thus, although several studies have addressed the secretion of stress and reproductive neuropeptides and the cellular activities of their respective endocrine neurons, none has yet characterized neuropeptide secretory responses for both the stress and reproductive axes together in a physiological preparation.
The sheep provides a model in which the secretory profiles and temporal association of hypothalamic hormones can be monitored with high resolution in conscious, normally behaving animals. Hypophyseal portal blood can be sampled at frequent intervals for prolonged time periods and, importantly, in sufficient volumes to monitor several hypothalamic neuropeptides in the same animal. In a recent study involving portal blood collection, we demonstrated that systemic endotoxin, a well characterized model of immune/inflammatory challenge (11, 12), inhibits pulsatile GnRH secretion in ovariectomized sheep, and this inhibition coincides with stimulation of peripheral cortisol (13). Earlier work in sheep demonstrated that systemic endotoxin markedly stimulates ACTH together with enhanced cortisol secretion (14). As both CRH and AVP are powerful stimulators of ACTH in sheep, either or both of these stress neuropeptides could have prompted activation of the adrenal axis. Here we tested the hypothesis that endotoxin stimulates CRH and AVP secretion into hypophyseal portal blood. In addition, we related the timing of the CRH and AVP responses to the previously characterized GnRH inhibition in these same animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the day before sampling, ewes were penned in pairs, and indwelling jugular catheters were installed. A calm, nonstressful sampling environment was created, minimizing human contact and maintaining natural light/dark cycles. Ewes were fed before sampling and were provided with hay and water during hypophyseal portal blood collection. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan.
Experimental design
Ewes were randomly assigned either to serve as controls (saline
vehicle, iv; n = 5) or to receive endotoxin (400 ng/kg, iv; n
= 5; Escherichia coli 05:B55, Sigma Chemical Co., St. Louis,
MO). Animals were sampled in pairs of one control animal and one
endotoxin-treated animal. Portal and jugular blood samples were
withdrawn continuously and dispensed into 0.5 ml ice-cold bacitracin
(3 x 10-3 M) at 10-min intervals for
14 h, beginning 4 h before and continuing for 10 h after
endotoxin or saline. Treatments were given between 1730–2000 h as
previously described (13). The dose of endotoxin (400 ng/kg, iv) was
based on a study in gonadectomized rams in which reproductive and
stress neuroendocrine alterations were induced, but severe adverse side
effects were avoided (14). Additionally, in pilot studies, we
established that this dose induced neuroendocrine alterations and
variable degrees of transient sickness behaviors in some ewes
(diarrhea, shivering, and lethargy). These behaviors were also observed
in the present study and generally subsided by the end of sampling.
After sample collection, ewes were killed with an overdose of
barbiturate (Beuthanasia, Schering-Plough Animal Health Corp.,
Kenilworth, NJ), and the pituitary was inspected to confirm appropriate
placement of the cut for sampling hypophyseal portal blood. Based on
careful visual inspection, it was confirmed that in no case did these
cuts include the pituitary stalk or posterior pituitary gland.
Assays
Within 1 h of collection, samples were centrifuged to
separate plasma from cells. An aliquot of portal plasma for GnRH assay
was immediately extracted with methanol. Separate aliquots for
subsequent CRH and AVP assays were snap-frozen and stored at -80 C.
All hormones were assayed in duplicate.
Assays for GnRH, LH, cortisol, and progesterone have been previously described in detail (13). Assay variation and sensitivities were as follows. GnRH intraassay variation (8 assays), as determined by the median variance ratio of assay replicates (17), averaged 0.062, and assay sensitivity averaged 0.23 pg/tube. LH intra- and interassay coefficients of variation (7 assays) were 7.8% and 7.7%, respectively, and assay sensitivity for 200 µl averaged 1.10 ng/ml. Cortisol intra- and interassay coefficients of variation (13 assays) were 15% and 9%, respectively, and assay sensitivity averaged 0.86 ng/ml. Progesterone intra- and interassay coefficients of variation (2 assays) were 9.7% and 7.9%, respectively, and assay sensitivity averaged 0.08 ng/ml.
Concentrations of CRH and AVP in acetone extracts of pituitary portal
plasma were assayed using antisera and protocols provided by Dr.
Charles Oliver (Marseilles, France). Both assays were previously
validated and used extensively for sheep pituitary portal plasma
(18, 19, 20, 21). Extracts were reconstituted in assay buffer (Tris-0.05
M HCl, 1% BSA, 0.1% Triton-X 100, and 0.1%
NaN3, pH 8.4) and assayed at 40–200 µl sample
equivalent/tube for CRH and 6.67–33 µl sample equivalent/tube for
AVP. For CRH assay, reconstituted plasma extracts were incubated with
antiserum to ovine CRH (100 µl; 1:30,000) at 4 C for 24 h
followed by [125I]CRH (10,000 cpm/100 µl; New England
Nuclear, Boston, MA) for 24 h. Antirabbit 
-globulin and
polyethylene glycol were used to separate bound and free CRH.
Assay sensitivity (95% confidence interval of buffer control) averaged
6.2 pg/tube; 50% displacement of [125I]CRH occurred at
39.4 pg/tube (CRH standard, Peninsula Laboratories, Belmont, CA). All
samples from a given ewe were measured in the same assay; the median
variance ratio of assay replicates averaged 0.074. The recovery of CRH
standard added to peripheral plasma before extraction averaged 84.2%;
all sample values were corrected for recovery assessed in each assay.
Validation of this previously described ovine CRH assay was confirmed
as follows. Procedural blank (value in extracts of peripheral plasma)
averaged 7.7 pg/tube. Blank values were determined in each assay and
subtracted from all sample values. Assay of increasing amounts of a
pool of portal plasma obtained during the peak response to endotoxin
caused a decrease in binding parallel to the standard curve when
corrected for procedural blank. The addition of peripheral plasma
extract to the standard curve did not alter its slope. CRH standard was
quantitatively recovered when added to peripheral plasma before
extraction and assay.
For AVP assay, antiserum (100 µl; 1:400,000) was incubated at 4 C with reconstituted plasma extracts for 24 h, followed by [125I]AVP (10,000 cpm/tube, New England Nuclear) for 24 h. Bound and free AVP were separated as described for the CRH assay. Assay sensitivity (95% confidence interval of buffer control) averaged 0.42 pg/tube; 50% displacement of [125I]AVP occurred at 2.2 pg/tube. All samples for a given ewe were measured in the same assay; the median variance ratio of assay replicates averaged 0.15. The recovery of AVP standard added to plasma before extraction averaged 60.2%; all samples were corrected for recovery assessed in each assay. Validation of this previously described ovine AVP assay was confirmed as follows. Procedural blank (value in extracts of peripheral plasma) was uniformly below assay sensitivity. Assay of increasing amounts of extract of pooled portal plasma obtained during the peak response to endotoxin caused decreased binding parallel to the standard curve. The addition of peripheral plasma extract to the standard curve did not alter its slope. AVP standard was quantitatively recovered when added to peripheral plasma before extraction and assay.
Data analysis
Hormonal values in portal blood were analyzed as collection rate
(picograms per min) rather than as concentration to minimize error due
to potential contamination of portal samples with peripheral blood or
cerebrospinal fluid (judged minimal by comparing portal and jugular
hematocrits) or due to changes in the flow of portal blood caused by
head position. Mean collection rates were calculated for each ewe
before and after endotoxin or saline injection and compared within
groups by paired t test. Thus, each ewe served as its own
control. Because CRH and AVP responses to endotoxin were immediate and
short lived, with values returning toward baseline levels by 4 h
after endotoxin treatment in most ewes, statistical analyses for these
hormones included data from 4 h before to 4 h after
treatment. The level of significance was set at P 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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depicts hypophyseal portal
CRH and AVP and peripheral cortisol for three representative control
ewes that received saline iv at time zero [cortisol data previously
reported (13)]. Comparing the 4 h before to the 4 h after
saline administration, no significant change was observed in either CRH
or AVP in portal blood; both hormones remained at or near assay
sensitivity. AVP in the peripheral blood was uniformly at assay
sensitivity (data not shown). Cortisol levels remained low throughout
sampling (generally <20 ng/ml) and exhibited a rhythmic, oscillatory
pattern unaffected by saline [values similar to those we observe in
ewes not undergoing portal sampling (13)]. Due to limitations in assay
sensitivity, temporal relationships between cortisol oscillations and
CRH and AVP could not be established.
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illustrates portal CRH and AVP,
peripheral cortisol, and core body temperature in three of the five
endotoxin-treated ewes. In contrast to saline vehicle, endotoxin
stimulated hypophyseal portal CRH and AVP. The maximal response for
both hormones occurred within 3 h of endotoxin, and for most ewes,
values were returning to baseline levels by 4–5 h after treatment. Of
note, the peak AVP response was much greater than that of CRH (mean
peak value; AVP, 423 ± 89 pg/min; CRH, 46 ± 10 pg/min). As
described previously (13), endotoxin significantly stimulated cortisol
secretion and core body temperature in these ewes.
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compares AVP patterns in
peripheral and portal blood of two representative endotoxin-treated
ewes. AVP is plotted as picograms per ml (rather than picograms per
min) to normalize portal values with AVP concentration in jugular
plasma. Overall, the AVP response to endotoxin was far greater in
portal blood than in jugular blood (note the logarithmic scale).
Although peripheral AVP did increase in all endotoxin-treated ewes,
this response was brief, much smaller, and generally delayed relative
to the massive stimulation of portal AVP. Of interest, both jugular and
portal AVP rose in parallel during the last few hours of sampling in
two of the five ewes (Fig. 3). This rise, however, was much smaller
than the massive increase in portal AVP observed during the first few
hours after endotoxin administration.
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depicts the time course of the
portal CRH and AVP stimulation in relation to previously reported (13)
values for portal GnRH, jugular LH, cortisol, and progesterone, and
body temperature in the two remaining ewes treated with endotoxin.
Before endotoxin administration, regular pulsatile discharges of GnRH
and LH typical of ovariectomized ewes (22, 23) were observed. Both
cortisol and progesterone remained basal. After endotoxin treatment,
GnRH and LH pulsatile secretion became suppressed coincident with
stimulation of portal CRH and AVP and peripheral cortisol and
progesterone. Of interest, in some ewes, GnRH and LH recovered from
endotoxin inhibition as CRH and AVP were decreasing (Fig. 4, ewe 6). In
others, however, GnRH and LH remained suppressed well after CRH and AVP
returned to baseline (Fig. 4, ewe 4). In the three endotoxin-treated
ewes not illustrated in Fig. 4, GnRH and LH pulsatile secretion were
also inhibited coincident with CRH and AVP stimulation (GnRH and LH
data in Ref. 13).
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).
Stimulation of these stress neuropeptides coincided with a significant
suppression of mean GnRH collection rate to approximately 30% of the
starting value (P < 0.05; Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By describing the time course of the CRH response and demonstrating concurrent AVP induction, our findings in sheep extend observations in the rat, showing that systemic IL-1 can increase CRH in portal blood (7). Interestingly, this study in rats did not reveal significant stimulation of hypophyseal portal AVP, although a large degree of individual animal variability was observed both before and after immune challenge. Such variability could be attributed to the acute surgical preparation and use of anesthesia, which may be stressors in and of themselves. Additionally, the collection technique used in rats required severing the pituitary stalk, thus damaging fibers of magnocellular neurons en route to the posterior pituitary and possibly increasing variability and obscuring the AVP release pattern. Neither of these caveats was at play in the present study, as we sampled conscious sheep and did not cut the pituitary stalk.
In line with the induction of AVP secretion in hypophyseal portal blood, endotoxin has been shown to stimulate a pronounced increase in cerebrospinal fluid levels of AVP in sheep (24). Further, a recent report by Dadoun et al. (25), which appeared while the present investigation was under review, indicates systemic endotoxin can stimulate both CRH and AVP secretion in portal blood of yearling rams (25). Additionally, other stressors, such as insulin-induced hypoglycemia (21, 26) and exposure to an audiovisual stress of a barking dog (26), can stimulate both CRH and AVP secretion in sheep. Of note in these studies, the AVP response was greater than that of CRH. In the present investigation, the peak AVP response in terms of picograms per min was approximately 10-fold higher than that of CRH. On a molar basis, the AVP response was even more massive because the CRH molecule is 4.5 times larger than that of AVP. Of interest in this regard, it has been suggested that AVP may be more potent than CRH in stimulating anterior pituitary ACTH secretion in the sheep (27).
Relative to the origin of AVP, two sources of this neuropeptide could contribute to its presence in portal blood: 1) first pass secretion into portal blood after release at the median eminence, and 2) recirculation through the portal vasculature after release into the periphery by the posterior pituitary gland. Several aspects of our findings support the former option. First, the portal AVP response was approximately 2 log-orders of magnitude greater than values in the peripheral blood. Second, in those ewes that exhibited a clear rise in peripheral AVP, the response tended to be delayed relative to the immediate massive stimulation of AVP in portal blood. Finally, the response in the periphery was very short lived, lasting for only a few 10-min samples in most ewes. In contrast, AVP in portal blood was elevated for several hours. Unlike our findings, the study of Dadoun et al. (25) mentioned above reported that endotoxin stimulates jugular AVP immediately and strongly. The small and inconsistent jugular AVP response we observed more conclusively suggests that AVP in portal blood is not magnocellular in origin. Supporting this conclusion, observations in the rat suggest that parvocellular neurons of the paraventricular nucleus can cosynthesize CRH and AVP (28).
Of interest, in some endotoxin-treated ewes, jugular AVP and portal AVP rose in parallel and in a quantitatively similar manner during the last few hours of sampling. This late AVP rise most likely reflects secretion into peripheral blood and thus a magnocellular source. It is possible that the late rise in AVP in the peripheral blood may be relevant to secondary activation of the neuroendocrine stress axis. In this regard, some effects of this immune challenge can persist for 24 h or more (29, 30). In the two ewes that exhibited a late peripheral AVP rise, cortisol did increase slightly late in sampling (ewes 4 and 6). Of note, however, other ewes that did not exhibit a late peripheral AVP rise also had elevated cortisol near the end of sampling (ewes 2 and 8). The possibility that a late rise in peripheral AVP is important for prolonged stress axis activation is interesting and worthy of future investigation.
The temporal relationships of the reproductive and stress neuroendocrine responses to endotoxin are also of interest. In all cases, GnRH suppression coincided with CRH and AVP stimulation. This temporal association provides a basis to consider whether the stress neuropeptides contribute to the suppression of pulsatile GnRH and LH secretion induced by immune challenge. Indeed, CRH and AVP can suppress tonic LH secretion in several species (31, 32), and CRH can inhibit GnRH release into portal blood on the afternoon of proestrus in the anesthetized rat (8). Do these neuropeptides, however, mediate reproductive neuroendocrine suppression induced by an immune/inflammatory-like challenge?
Studies in rodents provide strong evidence that at least CRH may not play a mediatory role, because lesion of the paraventricular nucleus and the central administration of CRH antagonists and antibodies do not prevent LH suppression induced by central cytokine challenge (5, 33, 34). In the ovariectomized monkey, however, antagonists to either CRH or AVP can reverse the suppression of LH pulsatile secretion induced by IL-1 given centrally, providing strong evidence that the stress neuropeptides do mediate reproductive neuroendocrine suppression in this primate species (35, 36). Although the roles of CRH and AVP in mediating the response to an immune challenge have not been addressed in sheep, it is of interest to note that CRH can actually stimulate LH pulsatile secretion in this species (37, 38). Further investigation is thus needed to determine whether the reproductive neuroendocrine suppression after endotoxin administration in sheep reflects the inhibitory actions of CRH and/or AVP, other hormones of the adrenal axis such as ACTH, cortisol, or progesterone, or whether GnRH suppression is independent of hypothalamo-pituitary adrenal activation. In this regard, it is important to emphasize that our studies address only CRH and AVP release at the level of the median eminence. Thus, the patterns we observe do not necessarily reflect the discharge of these neuropeptides in other brain regions that regulate GnRH secretion.
Finally, on a technical note, it should be emphasized that studies addressing the dynamics of hypothalamo-pituitary-adrenal function must always contend with the extent to which the experimental manipulations, in this study hypophyseal portal blood collection, may stress the animals in and of themselves. Although our procedures undoubtedly caused some disturbance, several observations suggest that they did not cause undue stress. First, control ewes exhibited clear rhythmic oscillations of cortisol, a pattern characteristic of basal glucocorticoid secretion in other species (39, 40). Second, the absolute levels of cortisol in the controls were similar to values we have observed in ewes not undergoing portal sampling (Ref. 13 and our additional unpublished observations). Finally, the animals were overtly calm, eating and drinking normally, and appeared undisturbed by our manipulations.
The foregoing observations permit the conclusion that a systemic immune/inflammatory-like challenge with endotoxin potently activates the neuroendocrine stress axis through central mechanisms, inducing marked and concurrent stimulation of both CRH and AVP secretion into hypophyseal portal blood of ovariectomized ewes. The induction of these stress neuropeptides coincides with the suppression of pulsatile GnRH release. Although our investigation does not directly address a functional link between CRH and AVP induction and GnRH inhibition, the temporal relationships characterized within the same animal provide a basis to pursue whether stress neuropeptide activation is causally linked to GnRH suppression after immune challenge in the sheep.
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Acknowledgments |
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Footnotes |
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2 Present address: Endocrinology and Metabolism Division, University
of Michigan Medical Center, 5560 MSRB II, Ann Arbor, Michigan
48109-1678.
Received February 2, 1998.
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References |
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mediates endotoxin induced suppression of
gonadotropin-releasing hormone pulse generator activity in the rat.
Endocr J 44:141–148[Medline]
on basal
gonadotopin release in the ovariectomized rhesus monkey: reversal by a
corticotropin-releasing factor antagonist. Endocrinology 128:2077–2082[Abstract]
-induced decrease in luteinizing hormone secretion
in the ovariectomized rhesus monkey. Endocrinology 131:153–158[Abstract]
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