Endocrinology Vol. 143, No. 1 117-129
Copyright © 2002 by The Endocrine Society
Intensive Direct Cavernous Sinus Sampling Identifies High-Frequency, Nearly Random Patterns of FSH Secretion in Ovariectomized Ewes: Combined Appraisal by RIA and Bioassay
Iain Clarke,
Lloyd Moore and
Johannes Veldhuis
Prince Henry’s Institute of Medical Research (I.C.), Clayton,
Victoria 3168, Australia; Wallaceville Animal Research Centre (L.M.),
Upper Hutt, New Zealand; and Endocrine Division, Department of
Internal Medicine, School of Medicine, University of Virginia (J.V.),
Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Prof. Iain J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail:
iain.clarke{at}med.monash.edu.au
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Abstract
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Analyses of FSH secretion suggest pulsatile, nonpulsatile, or
compositely pulsatile and nonpulsatile release modes. This may reflect
the reduced signal-to-noise ratio inherent in FSH pulse estimation
procedures and/or immunological-biological assay inconsistencies. To
address these issues, we sampled cavernous sinus and jugular venous
blood concomitantly from ovariectomized sheep at either 5-min or 1-min
intervals. Samples from the former were assayed by RIA, and those from
the latter by RIA and bioassay. Waveform-independent peak detection
revealed FSH pulses occurring at high frequency. Pulsatile FSH
secretion accounted for 28% of total secretion. Approximate entropy
analysis showed that FSH secretion was nearly random. There was
synchronous release of LH and FSH, but most FSH secretion was not
associated with LH release; 13% of discrete FSH and LH pulses were
concordant. We infer that FSH secretion exhibits pulsatile and
basal/nonpulsatile features, with high-entropy features. Linear and
nonlinear statistical measures revealed joint sample-by-sample
synchrony of FSH and LH release, indicating pattern coordination
despite sparse synchrony of pulses. We postulate that pattern synchrony
of FSH and LH release is effected at the level of the gonadotrope.
Concordant FSH and LH pulses probably result from pulsatile GnRH input,
but other mechanisms could account for independent FSH pulses.
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Introduction
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SYNTHESIS AND SECRETION of the
gonadotropins, LH and FSH, depends upon the pulsatile input of GnRH to
the gonadotrope. Thus, when the gonadotrope is deprived of GnRH, the
synthesis of the gonadotropin subunits and the secretion of the
gonadotropins is reduced (1). This primary secretagogue
regulates the secretion of the two gonadotropins differently, however,
because the withdrawal of pulsatile GnRH input leads to the immediate
cessation of the pulsatile secretion of LH, whereas FSH secretion
continues for some time (2). This distinction has prompted
the notion that there is a basal, or tonic, secretion of FSH, which is
not dependent on the secretagogue function of GnRH (2).
Following GnRH withdrawal, FSH secretion persists at a diminishing rate
over time, and for this reason (among others), it has been proposed
that FSH secretion is a direct reflection of the releasable pool of FSH
in the gonadotrope. According to this perspective, the secretion of FSH
does not require a pulsatile GnRH stimulus. Whether FSH secretion is
pulsatile and is directly linked to GnRH secretory pulses has been
addressed in various ways. Padmanabhan et al.
(3) collected blood from the anterior face of the
pituitary gland, which had been lesioned for the purpose of collecting
hypophyseal portal blood. They found that 93% of GnRH pulses were
associated with secretory pulses of FSH. This method of blood
collection from the lesion site would provide an admixture of blood
that was en passage to the pituitary gland and blood from
the sinuses of the secondary capillary bed of the portal system within
the pituitary gland. At this sampling site, and in some animals, plasma
FSH levels were up to 10 times higher than in the jugular vein plasma.
The method was taken to provide a reflection of the secretion of FSH
from the gonadotropes, but alterations in blood flow (due to the
opening of the pituitary gland at the site of the lesion) and admixture
of blood from two sources might have confounded facile interpretation
of the results. Using sampling of pituitary venous blood in the mare,
Irvine and Alexander (4) reported that the majority of
GnRH, LH, and FSH pulses were concurrent and concluded that GnRH is the
major secretagogue for both gonadotropins. Although these methods have
provided valuable information on the secretory profiles of LH and FSH
secretion, the issue of joint secretion of LH and FSH remains a matter
of some controversy, and we now examine the issue by sampling as close
as possible to the normal point of secretion from the pituitary gland,
namely the cavernous sinus. We have determined the patterns of
secretion of LH and FSH at this level and compared levels in the
cavernous sinus to those in the jugular vein by RIA and bioassay
(BIO).
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Materials and Methods
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Animals and ethics
Corriedale ewes, which had been ovariectomized (OVX) for at
least 1 month, were used during the time of the year when normal
ovary-intact animals were mating. The experiment was carried out
according to the guidelines for the Care and Maintenance of
Experimental Animals and was approved by the Monash Animal Ethics
Committee and the Animal Ethics Committee of The Victorian Institute of
Animal Science before commencement.
Surgery
Placement of a cannula into the cavernous sinus was achieved by
using a transnasal, transsphenoidal approach to the anterior face of
the pituitary gland as previously described (5), with
exposure of the cavernous sinuses anterior to the gland. The cavernous
sinuses are lateral to the pituitary fossa and extend anteriorly
(6). In this species, the carotid arteries enter the
sinuses and become the carotid rete (a dense plexus of fine blood
vessels) that lie within the sinuses (7). Ascending blood
then exits the rete through a common carotid artery (7).
The cavernous sinuses collect venous blood from the brain via the
transverse sinuses and from other venous outflow of the head,
e.g. the facial vein, which passes into the jugular veins
(6). The pituitary gland secretes into the cavernous
sinuses by passage of blood from the gland via the medial walls of the
sinuses. The medial wall of one cavernous sinus was punctured anterior
to the body of the pituitary gland with an 18-gauge hypodermic
needle and a cannula of polyethylene (internal diameter, 1.57 mm;
external diameter, 2.08 mm; WF Scientific, Huntingdale, Victoria,
Australia) was inserted into the sinus to a depth of 5 mm. The cannula
was advanced so that it lay alongside the wall of the sinus closest to
the pituitary gland, and care was taken not to rupture the carotid
rete. The entry point to the sinus was packed with GelFoam (Upjohn Pty
Ltd., Rydalmere, New South Wales, Australia), and the cannula was
secured within the operative tunnel in the sphenoid bone by dental
acrylic (Dentsply Ltd., Weybridge, Surrey, UK) introduced by syringe.
The cannula was then laid along the ethmoid plate and exited through a
burr hole in the nasal bone. It was passed sc to the back of the skull,
tied to the animal’s back, and closed with a three-way tap. The
cannula was kept patent with heparinized (50 U/ml) normal saline until
used (1–3 d later). The animal was allowed to recover for 2–4 d, and
on the day before sampling, a jugular venous cannula was inserted
(DwellCath; Tuta Laboratories, Pty Ltd., Lane Cove, New South Wales,
Australia) and connected to a manometer line that was also secured on
the animal’s back (as described above).
Animals were sampled one at a time, and two operators withdrew
simultaneous blood samples (2 ml) from the cavernous sinus and the
jugular vein. The blood was immediately centrifuged at 4 C, and the
plasma was stored at -18 C until assayed. Six sheep were sampled at
5-min intervals over 6 h in the first part of the study. In four
additional sheep, 1 ml paired blood samples were taken at 1-min
intervals over 4 h for both RIA and BIO of FSH. In the former
animals, blood volumes allowed measurement of LH in both cavernous and
jugular plasma. In the latter, LH assays were performed only on the
jugular samples (to define the position of the secretory pulses), and
the cavernous samples were devoted to the dual assay of FSH. There was
insufficient plasma for the assay of LH in the cavernous samples taken
at 1 min.
RIA of LH and FSH
For the samples in Exp 1, plasma levels of LH were assayed in
duplicate (100 µl) using the method of Lee et al.
(8). The standard used was ovine NIH-oLH-S18. For 12
assays, the average sensitivity was 0.1 ng/ml, the intra-assay
coefficient of variation (CV) was less than 10% over the range of
0.9–24 ng/ml, and the interassay CV was 10%. Plasma levels of FSH in
the samples in Exp 1 were determined by RIA as previously described
(9) with the ovine standard NIAMMD oFSH-RP-1. The
sensitivity of the assay was 0.2 ng/ml, the intra-assay CV was less
than 10% between 0.5 and 33 ng/ml, and the interassay CV was 23%.
Because the samples of Exp 2 were analyzed in a different laboratory
than those of the first, a different RIA was used (9). The
RIA was similar to the assay used for Exp 1 and was performed as
previously described with the exception that the iodinated FSH was
purified by ion exchange chromatography (10). The RIA used
USDA-oFSH-19-SIAFP-I-2 as the iodination reagent and NIAMMD-anti-oFSH-1
as the antiserum. The minimum detectable concentration of FSH was 0.2
ng/ml (USDA-oFSH-19-SIAFP-RP2) as defined by the concentration 2x
SD from the zero standard. The mean intra- and interassay
CV were 9% and 7%, respectively.
FSH BIO
The in vitro bioactivity was determined by examining
the ability of FSH to stimulate cAMP production by cultured Chinese
hamster ovary cells that express recombinant human FSH receptors
(11). The cAMP RIA was performed as described
(12). The assay had a sensitivity of 0.7 ng of FSH/ml
(USDA-oFSH-19-SIAFP-RP2) and an ED50 of 76 ng of
FSH/ml. The intra- and interassay CVs were 12% and 13%,
respectively.
Statistical procedures for two-hormone synchrony analysis
Three mathematically independent statistical approaches were
applied to appraise bivariate LH-FSH synchrony, cross-approximate
entropy (X-ApEn), cross-correlation analysis, and discrete peak
coincidence detection (13). Information gained is
complementary and nonredundant, because: a) X-ApEn quantifies
lag-independent and nonlinear synchrony of bihormonal release
(14); b) cross-correlation analysis detects lag-specific
linear relationships between successively paired sample measurements;
and c) discrete peak coincidence testing requires a priori
pulse identification followed by statistical concordance analysis
(13, 15). The complementarity of these analytical tools
have been reviewed recently (13, 14).
ApEn
Univariate ApEn comprises a class of model- and
scale-independent irregularity statistics designed to monitor the
relative orderliness of patterns in individual time series (16, 17). ApEn quantifies the subpattern reproducibility of
successive measurements and, thus, differs from pulse-detection
algorithms (14, 16, 17, 18). ApEn is a single non-negative
number, which provides an ensemble estimate of the regularity of any
given sequence. Higher ApEn denotes greater disorderliness or
randomness of patterns. Technically, ApEn quantifies the logarithmic
likelihood that patterns recur in the data (18, 19, 20).
ApEn is a family of three-parameter statistics, with members defined by
N, m, and r (below). For any given
data series containing N observations, two input parameters,
namely m and r, are defined, where m
represents the pattern (or window) length and r denotes the
tolerance (or threshold) for detecting pattern recurrence. To maintain
scale invariance, normalized r is defined as a percentage of
the between-sample SD of each time series
(e.g. 20%), and m is assigned a value of 1 or 2,
designating consecutive vectors of length 1 or 2 data points,
respectively. For the present time series, we calculated ApEn based on
r = 20% and m = 1, which is
abbreviated as ApEn (1,20%). This parameter set provides a sensitive
and replicable ApEn statistic, which has been validated for hormonal
time series of this length (14, 16, 21, 22, 23).
X-ApEn
To quantify joint synchrony for paired time series, we used
X-ApEn (14 and see definition 5 in Ref. 24).
This statistic compares the conditional regularity of bivariate data
sets. X-ApEn is analogous to ApEn, except that calculations are
performed on the standardized (z score-transformed) sequences. In the
present study, we applied X-ApEn using m = 1 and
r = 0.2, which ensures good statistical replicability
for the time-series lengths studied here. Further mathematical
discussion of X-ApEn, and a comparison with bivariate spectral and
cross-correlation assessments, is given in the appendix of Pincus
et al. (14).
Error estimates for ApEn and X-ApEn
Empirically based error estimates for individual ApEn or X-ApEn
values were made via Monte Carlo simulations (25).
Each simulation consisted of 1,000 random shufflings of the original
individual or paired data series without replacement to estimate a mean
"random" (as distinguished from "observed") ApEn or X-ApEn. The
resultant "null distribution" for each (paired) time series was
used to estimate the mean, SD, and maximal value of random
ApEn and X-ApEns. Maximally random was defined as the highest single
ApEn or X-ApEn value generated in the 1000 shuffled series. The ratio
of the observed (actual) to random (shuffled) ApEn (and the
SD of this ratio) was computed analogously by 1,000
reassortments of each data series. This ratio would tend to normalize
ApEn comparisons between hormones measured in different assays or based
on sequences of different lengths.
Statistical comparisons
Statistical contrasts in ApEn ratio values for the two hormones
were evaluated via a paired two-tailed unequal-variance t
test and were confirmed nonparametrically by the Wilcoxon (Mann-Whitney
U) statistic.
Cross-correlation analysis
Cross-correlation analysis appraises the linear (Pearson’s)
correlation coefficient between successively paired measurements in two
time series of equal length and spacing considered simultaneously (zero
lag) and at various other (nonzero) lag times (13). For
example, LH is compared with FSH measured concurrently (zero lag), one
sample later (or unit sample lag, here 5 min), or one sample earlier. A
positive lag interval was defined here as a change in LH preceding that
of FSH by that particular lag time (and, vice versa, for a negative
lag). Significant r values (cross-correlation coefficients)
at any given time lag were defined analytically based on the
corresponding within-sample pooled SD values
adjusted for series length and the number of lag units tested
(13). The overall significance of a group of r
values at any given lag time was examined via the Kolmogorov-Smirnov
statistic. Thereby, we test the null hypothesis that the corresponding
z score distribution has a zero mean and unit SD.
Because multiple comparisons are involved in cross-correlation
analysis, statistical significance was construed at the conservative
threshold of P < 0.01. This restricts the type I
statistical error to an asymptotic mean of 1 per 100 comparisons.
Discrete peak coincidence testing
Cluster analysis was applied as a model-free and
baseline-independent technique to detect discrete peaks in the paired
LH and FSH time series (26). We used t
statistics of 2.0 to identify significant two-point upstrokes and
two-point downstrokes in the 5-min data, and at a statistic of 3.0 with
three-point test nadirs and peaks for 1-min data, in both cases based
on within-series concentration-dependent pooled sample variances
(27). Cluster analysis thereby enumerates peak number;
interpeak interval length; and maximal (absolute values), fractional
(percentage), and incremental peak amplitudes over the preceding nadir.
The interpeak interval is computed as the time between successive peak
maxima.
Pulse coincidence was defined by the simultaneous or time-lagged
concordance of peak maxima in the paired series. The hypergeometric
(joint binomial) probability density function was applied to calculate
the expected random pulse concordance rate and to test the null
hypothesis that the number of observed peak coincidences reflects
chance associations alone, conditional on the number of peaks
identified in each of the paired series and the total series length.
Group coincidence probability values were approximated assuming
concatenation (13, 15, 28, 29).
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Results
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Five-minute sampling: RIA
As shown in Fig. 1
, catheter
placement was corroborated by the significantly
(P < 0.0001) higher levels of LH found in cavernous
sinus plasma compared with levels in the jugular. There was a smaller
(1.3-fold) but significant (P = 0.0025) differential
for plasma FSH.
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Figure 1. Matching plasma concentrations of LH and FSH in
cavernous sinus (left column) and jugular venous
(right column) blood samples from six individual ewes
(A–F). Samples were collected simultaneously from each site at 5-min
intervals (see Materials and Methods for details).
Error bars represent the predicted assay sample
SD values, and vertical arrows denote
identified pulses.
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By discrete peak detection analysis, the median LH interpulse intervals
in the cavernous sinus and jugular veins were, respectively, 43 and 50
min (P = NS; Fig. 2A).
Median (as well as mean ± SEM) interpeak
FSH intervals (min) were also comparable statistically at the two
sampling locations, 39 in cavernous and 70 in jugular
(P = NS; Fig. 2A). Thus, LH and FSH pulse frequencies
were not distinguishable (except for higher variance for FSH
pulsatility) in sheep based on a 5-min sampling paradigm.
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Figure 2. Discrete peak-detection (cluster) analysis of
5-min LH and FSH release profiles sampled in the cavernous sinus and
internal jugular veins. The upper panel gives the
individual data values of the interpeak intervals (min) with the group
mean ± SEM. The lower panel depicts
fractional peak heights (percentage increase of the peak maximum over
the preceding nadir, with the latter defined as 100%). Values of
P denote results of ANOVA on the logarithmically
transformed values (NS = P > 0.05).
Unshared alphabetical superscripts define statistically
contrasting means.
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Secretory peak heights, expressed as percentage of increase over
preceding nadir (100%), were substantially higher for LH in cavernous
than jugular blood; i.e. the median was 731 for cavernous
and 227 for jugular (P = 0.0006) samples (Fig. 2B
).
This 5-fold ratio for LH contrasted with a nonsignificant difference
for detectable FSH peak heights; the median was 142 for cavernous and
130 for jugular samples. Thus, compared with LH, FSH peak heights were
8.5-fold lower in cavernous blood and 4-fold lower in jugular blood.
The estimated percentage of pulsatile secretion (sum of peak
area/basal) was, for LH, 63 ± 5% (cavernous) vs.
38 ± 4% [jugular (P = 0.0074)]. Comparable
figures for FSH were 28 ± 8% (cavernous) vs. 20
± 3% [jugular (P = 0.0015; Fig. 3]. These prominent distinctions are
illustrated in Fig. 1.
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Figure 3. Mean (±SEM) percentage of total LH
and FSH release due to pulsatile secretion in time series from animals
sampled at 5-min intervals. Bars with arrows connect
post hoc-paired t test comparisons at the
indicated P values. Overall contrasts among the four
groups were evident at P = 0.01 (ANOVA).
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The ApEn statistic was used to quantify the relative orderliness of LH
and FSH secretory patterns (see Materials and Methods and
Fig. 4). LH secretion into cavernous
blood was significantly more orderly than that into jugular blood;
i.e. the median LH ApEn ratio was 0.512 for cavernous
vs. 0.794 for jugular samples (P = 0.017).
Lower ApEn ratios denote more regular patterns in the data, and a ratio
of 1.0 denotes a mean random pattern (equivalent to shuffled data
series). In contrast, the median FSH ApEn ratios approached unity for
cavernous (0.948) and jugular (1.015) values (P = NS).
Indeed, jugular FSH fluctuations were statistically indistinguishable
from those of randomly shuffled data series (mean random FSH ApEn ratio
of 1.000 ± 0.0385). The foregoing inferences were corroborated by
expressing LH and FSH ApEn values as SD values
removed from the mean value of 1,000 randomly shuffled series (Fig. 4, lower panel). LH ApEn in cavernous blood was 7.4 ±
0.73 SD values removed from the mean random
(P < 10-6), but FSH ApEn was
only 1 ± 0.45 SD values from the mean
random (P = 0.86).
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Figure 4. ApEn ratios (upper panel) and ApEn
SD values from the mean random (lower panel)
for the indicated sampling sites (jugular or cavernous sinus) and
hormones (LH and FSH). The ApEn ratio defines the mean ratio of the
individually observed ApEn value to that of 1,000 randomly shuffled
renditions of the same time series. The number of SD values
by which the observed series ApEn is removed from mean random
(shuffled) ApEn provides a complementary measure of relative
orderliness. An ApEn ratio of unity and a number of SD
values from mean random of zero approach mean random expectation.
Accordingly, LH release monitored every 5 min in the cavernous sinus is
significantly orderly, whereas FSH patterns in jugular blood are not
distinguishable from randomly shuffled data (see Materials and
Methods). Unshared alphabetical superscripts
define statistically contrasting means Arrows connect
post hoc-paired t test comparisons at the
indicated P values. Overall contrasts among the four
groups were evident at P = 0.01 (ANOVA).
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Synchrony of LH and FSH release was quantified by complementary linear
cross-correlation, nonlinear (pattern-sensitive) X-ApEn, and
discrete peak coincidence probability analysis. Cross-correlation
analysis established strong synchrony of LH release observed at the two
different sampling sites at a zero or 5-min time lag only
(P < 104 for both), and between
LH and FSH in the cavernous sinus at zero time lag (P
< 10-4; Fig. 5). In contrast, FSH secretion
patterns in the cavernous and jugular sites were independent, as was
FSH and LH as determined at the jugular site (data not shown).
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Figure 5. Cross-correlation coefficients (y-axis,
r values) are given as the median and absolute range for
the indicated paired hormone time series evaluated simultaneously and
at 5-min intervals lag times (x-axis min) and denote the sampling
intervals separating observed associations at the indicated group
significance P value (see Materials and
Methods). The upper panel compares LH
secretion determined from cavernous and jugular samples. When zero lag
was observed (coincident peaks) or when the peak occurred 5 min earlier
(lag -5) in the cavernous sample, highly significant
cross-correlations were obtained. The lower panel shows
cross-correlation for LH and FSH secretion as observed in the cavernous
samples, with zero lag indicating coincidence of secretion around the
time (-20 min to +20 min) of identified LH pulses.
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Nonlinear X-ApEn demonstrated complete loss of LH-FSH pattern synchrony
in the jugular vein data from the 5-min sampling series (data not
shown). There was, however, significant pattern coordination (compared
with randomly shuffled LH and FSH pairs) in the data derived from the
samples taken at 5-min intervals from the cavernous sinus
(P < 10-3; data not shown).
Using data from the 1-min sampling (cavernous sinus), FSH RIA and FSH
BIO showed detectable (nonrandom) orderliness (P <
0.05 for both cases) but remarkably less regularity than the LH taken
from the jugular samples (P = 0.00011; Fig. 6). FSH values from either assay
maintained significant (P < 0.025) nonrandom synchrony
with LH values in the jugular samples (1-min sampling) (Fig. 6).
Discrete peak coincidence testing via the hypergeometric probability
density revealed significant group (total) LH/LH peak concordance at
the cavernous and jugular sites (P <
10-8) and nonrandom total LH/FSH peak
concordance among the groups of animals at the cavernous site only
(P = 0.042; Fig. 7).
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Figure 6. ApEn data for 1-min sampling of cavernous sinus
FSH release as quantified by RIA or BIO and LH secretion as measured by
RIA. Data are presented exactly as described in the legend of
Fig. 4.
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Figure 7. Total number of observed
(asterisks) and randomly expected (horizontal
dashed lines) coincident peaks for the indicated hormone pairs
in the entire group of animals studied. Values of P
denote the probability that the observed rate of peak concordance
reflects chance associations alone.
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One-minute sampling: RIA and BIO
Data obtained from 1-min sampling are illustrated for two animals
in Fig. 8. Mean FSH concentrations by RIA
and BIO in the cavernous sinus were 5.0 ± 2.0 (median = 5.0)
and 11.5 ± 2.1 (median = 11.8), respectively
(P = 0.017). The median interpeak interval (min) was
12.5 by RIA and 10.8 by BIO (NS; Fig. 9, upper panel). Fractional peak amplitudes (percentage) for
FSH measured every 1 min in the cavernous sinus were (median) 143 by
RIA and 214 by BIO (P = 0.013; Fig. 9, lower
panel). LH interpulse intervals and percentage peak heights were
analogous in the 1-min and 5-min data (Fig. 9 and above), providing a
suitable control. Figure 10 shows the
lack of significant (nonrandom) X-ApEn between 5-min jugular LH and
5-min jugular or cavernous FSH series.
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Figure 8. Plasma LH (jugular) and FSH RIA or BIO (cavernous
sinus) concentration profiles observed by sampling at 1-min intervals
from the cavernous sinus in two ewes. Error bars
represent the predicted assay sample SD values.
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Regularity of the 1-min FSH secretory profiles (Fig. 8) was assessed by
the ApEn ratio of observed to random (shuffled series). FSH ApEn ratios
were similar in both assay systems; median = 0.945 (RIA) or 0.948
(BIO) [NS]. Both values were more irregular than that for LH
(median = 0.655; P = 0.00011). Likewise, FSH ApEn
ratios were only minimally removed from random expectation (mean ApEn
of 1,000 shufflings of each original FSH time series). The observed
ApEn values for FSH were removed from mean random by 2.33 ± 0.99
SD values (P < 0.01) for RIA and
1.74 ± 1.1 SD values for BIO (NS). In
contrast, 1-min LH secretion was highly ordered based on an LH ApEn
ratio that was removed from random by 14.5 ± 1.6
SD values (P <
10-6). Cross-correlation of FSH RIA and BIO time
series was significant (P < 0.01) in three of the four
individual animals (maximal z scores +2.3 to +4.1). FSH RIA/BIO X-ApEn
for 1-min cavernous data averaged 0.956 ± 0.0250
(P < 0.01 vs. random). FSH RIA and BIO
pulses tended toward nonrandom discrete-peak concordance
(P = 0.11).
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Discussion
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High-intensity sampling from the point of secretion (cavernous
sinus) in adult OVX sheep and detailed biomathematical analyses of
pituitary FSH secretion unveiled several novel features of the
pulsatile and entropic (pattern-sensitive) control of this hormone. By
concomitant LH measurements, we showed nonrandom joint synchrony of FSH
and LH release in this model. Validation experiments based on 1-min
cavernous-sinus sampling data and combined RIA and BIO of the FSH time
series further disclosed unexpected high frequency oscillations of FSH
and established that the subordinate patterns of FSH release were
minimally ordered. Collectively, these data thus illuminate both common
and unique properties of FSH and LH neuroregulation in vivo
(vide infra).
Pulsatile analyses identified consistently episodic release of FSH in
both the 5- and 1-min sampling paradigms, as corroborated and by both
RIA and BIO in a total of 10 animals. The mean (fractional) FSH pulse
amplitude ranged from 145 to 214% (RIA, BIO, and both 5- and 1-min
data). The lower mean amplitude of 145% exceeds nominal FSH assay
imprecision by 4- to 6-fold. Moreover, in the 5-min data set, when LH
was assayed simultaneously in cavernous blood, FSH/LH pulse concordance
was markedly nonrandom. Whereas FSH pulsatility was statistically
unequivocal, two particular features distinguished FSH pulses from
those of LH. Firstly, the relative amplitude of LH pulses was 8.5-fold
higher than that of FSH pulses at the same sampling site, and secondly,
there were unanticipated high-frequency FSH oscillations superimposed
in 1-min sampled RIA and BIO FSH time series.
Although mean FSH and LH interpulse intervals (reciprocal of pulse
frequencies) were indistinguishable statistically based on a 5-min
sampling paradigm (i.e. 42 ± 4.7 min for FSH and
45 ± 1.7 min for LH), FSH oscillations recurred every 11–13 min
(RIA and BIO, respectively) in the 1-min time series. When FSH was
measured by BIO, the mean amplitude of these rapid pulsations was
214 ± 8.7%. Hormonal oscillations of this high frequency have
been recognized previously for GH (based on 30-sec blood sampling; Ref.
30), PTH (31), insulin (based on 1-min blood
sampling; Ref. 32), and possibly LH and FSH in
postmenopausal women (27, 33). Except for the present
direct cavernous sinus blood sampling protocol, however, other studies
relied upon measurements in peripheral/systemic blood wherein the ratio
of the signal amplitude to the basal hormone level is markedly damped.
Indeed, in the present study, the relative amplitude of LH pulses
evaluated at the cavernous vs. jugular sampling sites
exceeded 9-fold and for FSH was approximately 1.5-fold. The modest
differential in FSH levels illustrates the prominent recirculation
properties of this hormone, which limits pulsatility resolution.
Complementary to pulse analysis is the quantification of subordinate
(nonpulsatile) patterns of ad seriatim hormone release
(14, 21, 22, 34, 35, 36). The latter regularity analysis
provides information about the complexity and relative strength of
input (feedback and feedforward) signals controlling moment-to-moment
hormone output, e.g. as quantified by ApEn
(25). Mean FSH ApEn values in 5-min and 1-min time series
were minimally distinguishable from random. Only FSH ApEn calculated
for 1-min FSH time series showed significantly nonrandom patterning,
because the observed ApEn ratios were 2.33 SD
values removed from the mean random ApEn (obtained by shuffling each of
the four data series 1,000 times). For LH secretory patterns in jugular
blood determined by either 5- or 1-min sampling, ApEn ratios were 7.4
and 14.5 SD values removed from mean random
expectation (P < 10-6 to
P < 10-12). Accordingly, the
present data establish unequivocal statistical contrasts in the
relative complexity of FSH and LH secretory control, whether measured
in the cavernous sinus or the jugular vein in the same animal. FSH ApEn
ratios tended to show less disorderliness in cavernous than in jugular
blood, in accordance with the earlier findings of Padmanabhan et
al. (3). On the other hand, Irvine and Alexander
(4) showed a very close relationship between GnRH, LH, and
FSH secretion when sampling cavernous sinus blood in the mare. We
sought to measure gonadotropin secretion at the point of secretion,
downstream from the gonadotrope and at the normal exit point from the
pituitary gland. In this way, our study hopefully provides data that
are representative of the normal physiological process. In comparison
with the data obtained in the horse (4), it seems likely
that there is a substantial species difference, such that the majority
of LH and FSH is secreted in synchronous pulses in the horse, whereas
the same is not true in the OVX ewe. The present analyses differ from
the previous reports (3, 4) by way of providing a 3-fold
complementary quantification of pairwise LH and FSH synchrony, namely
cross-correlation, X-ApEn, and discrete peak coincidence probability
analysis. Furthermore, with respect to FSH, comparable inferences were
made by RIA and BIO. Although agreement among these methodologies
offers an important basis for our inferences, comparably comprehensive
assessments of data collected in other laboratories and in additional
animals will be important to extend these findings.
The highly irregular (virtually random) subpatterns of FSH release, but
not LH release, provide unequivocal evidence of unequal control of
these two hormones but do not establish the precise cellular or
neuromodulatory mechanisms that drive this distinction. Indeed,
analogous dissociation between the relative orderliness of FSH (highly
irregular) and LH (more orderly) is evident in the human but vanishes
with increasing age in both women and men (37, 38). Highly
irregular subpatterns of (nonpulsatile) hormone secretion are also
prominent in neuroendocrine tumoral states, such as acromegaly,
Cushing’s disease, and prolactinoma (34, 39, 40). Tumoral
conditions further emulate FSH output patterns in their predominance of
nonpulsatile release; only 28% of cavernous FSH secretion was
pulsatile vs. 63% for LH in the same animals. These
features suggest that FSH production is driven at a high basal
nonpulsatile level. The high ApEn points further to a composite of
interactive factors, which jointly confer a highly irregular release
process. Plausible candidate regulators include intrapituitary activin,
follistatin, and inhibin; extrahypophyseal, adrenogonadal signals
(e.g. sex-steroid hormones); and other hypothalamic inputs
(e.g. GnRH and other possible modulators of FSH
release or inhibition).
Concomitant measurements of cavernous-sinus FSH and LH secretion at
5-min intervals allowed us to appraise coordinate secretion of these
two gonadotropins. We used three complementary strategies. First, via
the linear estimation procedure of cross-correlation analysis, we
identified highly significant (P <
10-4) covariations in sample-by-sample FSH and
LH release. Secondly, the analytically independent X-ApEn statistic
delineated nonrandom synchrony of the patterns of FSH and LH secretion.
This finding was unexpected, given the near-randomness of FSH release
considered alone. Indeed, this observation unmasks quantifiable
(P < 0.01 vs. random) pattern-specific
coupling between the basal/nonpulsatile control of these two hormones.
Thirdly, discrete FSH and LH peak-coincidence analysis established
nonrandom co-pulsatility (P = 0.042). Albeit
statistically significant, concordant FSH and LH pulses constituted the
minority of events (i.e. 13% observed vs. 5%
expected on the basis of change alone). Accordingly, the foregoing
perspectives provide evidence for both common (cross-correlation and
X-ApEn) and distinct (peak discordance rate of 87%) mechanisms of FSH
and LH neuroregulation. These data agree with those of Padmanabhan
et al. (3) but do not show the vast discrepancy
between FSH levels at the point of secretion and in jugular blood. We
conclude that the majority of FSH secretion is not associated with
GnRH/LH pulses.
The present data provide a platform for the examination of upstream and
downstream implications of high frequency pulsatile secretion of FSH.
We speculate that the latter derives from local intrapituitary
regulation of FSH secretion by autocrine and paracrine factors. This
may involve localized cell clusters being acted upon by such factors.
Mathematical modeling would support the plausibility of this
conjecture, but studies of the intact pituitary gland and/or regions of
gonadotrope cells in normal clusters would be required to affirm or
refute this notion. In contrast, the mechanism of LH release, based on
extensive studies with GnRH antisera or antagonist peptides (41, 42, 43), appears to be exclusively dependent upon the pulsatile
secretion of GnRH from the hypothalamus. In essence, these conclusions
are in broad agreement with Padmanabhan et al.
(3). Fewer data are available to appraise the downstream
effects of pulsatile LH and FSH secretion on gonadal target cells. In
some studies, episodic stimulation drives biochemical responses more
effectively, and in others the reverse is true (27, 33).
In summary, the present experimental analyses combine highly intensive,
central cavernous-sinus blood sampling with RIA and BIO of FSH to
appraise the regulation of in vivo FSH secretion in the
gonadectomized sheep. Thereby, we delineate unequivocal FSH
pulsatility, but at 8.5-fold lower relative (fractional) mean amplitude
than that of LH. FSH is secreted primarily in a basal/nonpulsatile mode
with essentially random patterning of this component. High sampling
density (1-min intervals) and sophisticated analysis has revealed rapid
oscillations in FSH release occurring every 11–13 min. Synchrony of
FSH and LH secretion points to sample-by-sample covariation of
regulatory processes. In contrast, albeit nonrandomly associated,
discrete pulses of FSH are discordant from those of LH in 87% of
cases, thus unveiling distinctions in pulse generation. In ensemble, we
infer from our data that both common and distinct neuroregulatory
mechanisms govern pituitary FSH and LH secretion in the OVX ewe.
Whether analogous complexity of bihormonal secretory control operates
in the gonad-intact animal is not known.
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Acknowledgments
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We thank Bruce Doughton and Karen Perkins for animal care and
assistance with the experiments and Alix Rao, Winny Ng Chie, and Wayne
Young for RIAs and BIOs.
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Footnotes
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This work was supported by The Australian National Health and Medical
Research Council NIH U54 Reproduction Center.
Abbreviations: ApEn, Approximate entropy; BIO, bioassay; CV,
coefficient of variation; OVX, ovariectomized; X-ApEn,
cross-approximate entropy.
Received June 29, 2001.
Accepted for publication October 17, 2001.
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Abstract
Introduction
