0013-7227/97/$03.00/0
1
Endocrinology
Copyright © 1997 by The Endocrine Society
Neuroendocrine Control of Follicle-Stimulating Hormone
(FSH) Secretion. I. Direct Evidence for Separate Episodic
and Basal Components of FSH Secretion*
VASANTHA PADMANABHAN, KRISTIN MCFADDEN, DAVID T. MAUGER†,
FRED J. KARSCH, AND A. REES MIDGLEY, JR.
Reproductive Sciences Program (V.P., K.M., F.J.K., A.R.M.) and the Departments of Pediatrics (V.P.),
Physiology (F.J.K.), and Biostatistics (D.T.M.), University of Michigan, Ann Arbor, Michigan
48109-0404
ABSTRACT
Continuous sampling of hypophyseal portal blood from unre-
strained sheep is providing an unprecedented means for measuring
and defining the characteristics of the secretory profile of GnRH. With
this method, GnRH has been shown to be released in discrete pulses
lasting 5– 8 min, with the amplitude of some pulses exceeding 50-fold.
Although the relationship between these pulses and the accompany-
ing pulses of LH measured in the jugular vein are unambiguous, the
relationship of GnRH pulses to the release of FSH has not been well
defined due to the longer clearance of FSH. In previous studies we
have shown that hypophyseal portal blood, in addition to serving as
a source material for hypothalamic secretions, provides a means to
define secretory patterns of pituitary hormones. Because of this we
hypothesized that the GnRH-FSH secretory relationships would be
easier to define in hypophyseal portal than in jugular vein blood
before the secretory products are subjected to dispersion and clear-
ance in circulation. To test this possibility, we monitored hormonal
patterns in blood collected at 5-min intervals for 6 –12 h from the
I N STRIKING contrast to the wealth of information avail-
able regarding LH secretion, our understanding of the
regulation of FSH is scanty. To a large degree, this is because
circulating patterns of FSH in peripheral blood are hard to
decipher due to the long circulating half-life and apparent
interpulse secretion of FSH. The available evidence suggests
that, in contrast to the dependence of the LH secretory system
on GnRH pulsatility (1), FSH secretion is regulated by a dual
mechanism, one controlling the basal and the other control-
ling the pulsatile component of FSH secretion (2– 6). The
difficulty in monitoring secretory profiles in vivo and the long
Received July 31, 1996.
Address all correspondence and requests for reprints to: Dr. Vasantha
Padmanabhan, Reproductive Sciences Program, 300 North Ingalls
Building, Room 1101, Ann Arbor, Michigan 48109-0404. E-mail
vasantha@umich.edu.
* This work was performed as part of the National Cooperative
Program for Infertility Research, was supported by NIH Grant U54-
HD-29184, used samples generated in an earlier study funded by NIH
Grant R01-HD-18018, and received the support of the Assay and Re-
agents, Sheep Research, and Biostatistics Cores of the Center for the
Study of Reproduction (NIH Grant P30-HD-18258). Portions of this work
were presented at the 74th Annual Meeting of The Endocrine Society,
San Antonio, Texas, 1992.
† Present address: Center for Biostatistics and Epidemiology, Penn-
sylvania State University College of Medicine, 500 University Drive,
Hershey, Pennsylvania 17033.
424
Vol. 138, No.
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peripheral and hypophyseal portal circulation of six ovariectomized
ewes from a previous study. In contrast to the nonpulsatile pattern
of FSH in the peripheral blood, 93% of the GnRH pulses were asso-
ciated with essentially coincident, discrete pulses of FSH in the portal
plasma. Of potentially even greater interest, additional episodes of
FSH release were clearly discernible between the GnRH-associated
pulses of FSH. As concentrations of peripheral plasma FSH did not
reach those in hypophyseal portal plasma, the inter-GnRH episodes
of FSH secretion could not result from contaminating peripheral
blood. In addition to the episodic mode of secretion, substantial
amounts of FSH were found between FSH pulses. This basal com-
ponent of FSH appeared to be the dominant mode of secretion rather
than pulses. The results of this study not only confirm that GnRH
pulses lead to pulsatile release of FSH, they also suggest that some
other mechanism or factor may be controlling the non-GnRH-asso-
ciated episodes as well as the basal components of FSH secretion.
(Endocrinology 138: 424 – 432, 1997)
circulating half-life of FSH precluded a definitive test of this
hypothesis.
Although several laboratories, including ours, have at-
tempted to characterize the pulsatile release of immunore-
active FSH from peripheral measurements in sheep and
other species, such assessments have often (7–10) not yielded
a convincing answer to a simple but fundamental question:
is FSH secretion episodic? The utilization of a surgical ap-
proach, which was originally established to characterize se-
cretory patterns of hypothalamic hormones (11), to define
secretory dynamics of pituitary hormones (12) has provided
us with a unique approach to address this question defini-
tively. The approach exploits the hypophyseal portal blood
collection technology (11, 13). Because hypophyseal portal
vessels are cut at the level of the pituitary, we postulated that
hypophyseal portal blood would serve as a suitable medium
for determining the secretory dynamics of pituitary secre-
tions. This premise proved to be true for LH (12). Capitalizing
on this approach, we here provide direct evidence that FSH
secretion in ovariectomized ewes is indeed comprised of
both episodic and basal components of release. Furthermore,
the episodic mode of FSH secretion appears to be comprised
of both GnRH-associated and non-GnRH-associated pulses
of secretion.
 FSH SECRETORY DYNAMICS
Materials and Methods
Experimental design
For determination of secretory patterns of FSH, blood samples col-
lected from the peripheral and hypophyseal portal circulations of ovari-
ectomized ewes from a previous study (14) were used. Details of the
surgical procedures, hypophyseal portal sample collection, and circu-
lating patterns of GnRH in hypophyseal portal blood and LH in both the
peripheral and hypophyseal portal circulations have been described
previously (12, 14). Briefly, adult Suffolk ewes were ovariectomized and
surgically fitted with an apparatus for collection of hypophyseal portal
blood, and the collection procedure was initiated approximately 1 week
later, using methods described previously (13). Integrated samples of
hypophyseal portal blood and jugular blood collected at 5-min intervals
for 6 h from five ovariectomized ewes (no. 1, 2, 5, 6, and 10) and for 12 h
from one ovariectomized ewe (no. 3) were used in this study. Samples
from ewes 1, 2, 6, and 10 were collected during the breeding season, and
those from ewes 3 and 5 were obtained during the anestrous season.
Hypophyseal portal samples were collected in tubes containing 0.5 ml
3 3 1023 m bacitracin in phosphate-buffered isotonic saline. FSH con-
centrations in both the peripheral and hypophyseal portal samples were
determined. Each series of portal and peripheral FSH measurements was
compared with corresponding measurements of previously reported
GnRH (14), peripheral LH (14), and hypophyseal portal LH (12).
All procedures were approved by the University Committee on the
Use and Care of Animals.
RIAs
Hypophyseal portal and jugular FSH were assayed in duplicate by a
previously validated RIA (15–17). The assay uses the 620 antibody at a
1:48,000 dilution (17) and purified ovine FSH (NIDDK oFSH-1) for
iodination as well as a reference standard. All samples from each animal
were measured in a single assay. The assay sensitivity (2 SD from the
buffer control) averaged 3 pg/tube (range, 2–5 pg). The cross-reactivity
of the a-subunit averaged less than 0.3%. Intra- and interassay coeffi-
cients of variation based on three quality control pools at approximately
0.9, 1.7, and 6.0 ng/ml averaged less than 12%.
To minimize degradation of GnRH during collection and to chill
hypophyseal portal samples in an ice bath as quickly as possible, the
collection rate was set at a higher speed than the rate of flow of hy-
pophyseal portal blood into the collection apparatus (13). This led to the
collection of hypophyseal portal blood as discrete blocks segmented by
air; this approach minimized dispersion of secreted products during
collection. Jugular blood, on the other hand, was collected as a contin-
uous stream in the collection line. To allow direct comparisons with
concentrations of FSH in peripheral samples (nanograms per ml), all
measurements of FSH (as well as LH and GnRH) in hypophyseal portal
plasma are reported as concentrations. Furthermore, to adjust for the
difference in transit time in the collection line (10 –12 min for jugular
blood and 3 min for hypophyseal portal blood), samples are offset by 10
min (two samples) for the purpose of plotting. It should be noted that
a small error of 1–3 min may remain.
Statistical analysis
The measured concentration of hormone in the hypophyseal portal
blood is the sum of that which is secreted plus that which recirculates.
To obtain an estimate of secreted FSH, the concentration of FSH in each
jugular sample was subtracted from that in the corresponding hypoph-
yseal portal sample. Although subtracting the jugular concentrations
adds another, small source of error to the portal data, the variation in
jugular concentrations is so much smaller than changes in hypophyseal
portal circulation that subtracting the jugular values is for all practical
purposes like subtracting a small constant. All hormonal series from
each ewe were analyzed with the Kushler-Brown Pulsefit algorithm (18).
This statistical model is nonlinear, assumes exponential decay, and
attempts to account for the error in measurements due to biological noise
as well as assay error. Pulses are identified by stepwise selection. As the
algorithm is limited to identifying pulses that are no longer than two
observations on the upslope, a postprocessor merges pulses that are
contiguous in time into a single pulse. Concordant pulses of GnRH and
FSH, LH and FSH, and hypophyseal portal FSH and jugular FSH were
425
identified; pulses beginning within 10 min (two samples) of each other
were considered to be concordant. The average lag time between con-
cordant pulses was also estimated for each ewe. In addition, to assess
the overall temporal relationships between hormone patterns, the cross-
correlation for the above variables was calculated at different time lags
(autocross-correlation). The time lag that yields the highest cross-cor-
relation is an estimate of the overall time lag between two series. The
average pulse lag time simply estimates the temporal relationship be-
tween concordant pulsatile episodes. Within-animal comparisons, such
as amplitude of GnRH-associated vs. non-GnRH-associated pulses, were
performed using paired t tests or the nonparametric Wilcoxon signed
rank test.
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Results
Figure 1 depicts representative patterns of hypophyseal
portal (top panel) and peripheral FSH in two ovariectomized
ewes sampled during the anestrous season (ewe 5 and 6 h in
ewe 3). Levels are plotted on the same scale to provide an
estimate of the magnitude of differences between the pe-
ripheral and hypophyseal portal levels. For comparison, pre-
viously reported hypophyseal portal and jugular patterns of
LH (14) are shown in the bottom panel. Concentrations of FSH
in hypophyseal portal blood were several-fold higher than
those in the peripheral circulation. FSH patterns in hypoph-
yseal portal blood showed a distinctively episodic pattern of
release that appeared to be superimposed over a basal level
of secretion. The relative increases in FSH secretory episodes
appeared to be lower in magnitude for FSH than LH. Fur-
thermore, the magnitude of changes occurring between FSH
in portal and jugular circulations often did not parallel the
changes occurring between hypophyseal portal and jugular
LH, even within a given sample.
Figure 2 shows patterns of hypophyseal portal (closed cir-
cles) and jugular FSH (open circles) in two other ewes sampled
during the breeding season (ewes 1 and 2). Also shown are
corresponding, previously reported patterns of hypophyseal
portal and jugular LH (lower panel) and GnRH (shaded patterns
in both panels). Comparison of time courses of GnRH, LH,
and FSH revealed that, although the dominant patterns of
GnRH and LH were pulsatile, the pattern of FSH was com-
prised of both pulsatile and basal components. In contrast to
the absolute one to one relationship of LH to GnRH (12, 14),
not all episodes of FSH release were associated with a de-
tectable GnRH pulse. Episodes of FSH secretory activity were
evident between GnRH-associated pulses of FSH. In several
instances (identified by arrows in Fig. 2), GnRH-associated
bursts of FSH appeared to develop upon a previously trig-
gered episode of FSH release.
Figures 3 and 4 summarize the patterns of secreted (see
Materials and Methods for calculation) and jugular FSH from
two ovariectomized ewes. To reveal more clearly the com-
parative magnitude of changes in the hypophyseal portal
and peripheral blood and to account for heterogeneity of
variance, values are plotted on a logarithmic scale. To pro-
vide an estimate of measurement errors, the range of dupli-
cate values is also shown (the thin lines running on either side
of the plotted concentrations of secreted FSH). Previously
reported patterns of GnRH (14) are coplotted to facilitate
comparisons. As reported previously (14), GnRH was re-
leased in discrete episodes, between which values returned
to an undetectable or nearly undetectable baseline. Each
pulse of GnRH was directly associated with a concomitant
426 FSH SECRETORY DYNAMICS Endo • 1997
Vol 138 • No 1

FIG. 1. Patterns of hypophyseal portal

and jugular FSH from two ovariecto-
mized ewes (no. 5 and 3) both sampled
during the anestrous season. Previ-

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ously reported patterns of hypophyseal
portal (12) and peripheral LH (14) are
coplotted for comparison. Statistically
identified pulses of FSH in the hypoph-
yseal portal and peripheral samples are
shown (* and Ç in hypophyseal portal
and peripheral blood, respectively).

FIG. 2. Patterns of hypophyseal portal and jugular FSH patterns from two ovariectomized ewes (no. 1 and 2), both sampled during the breeding
season. Hypophyseal portal LH and peripheral LH patterns (14) from a previous study are provided in the lower panels for comparison. To
understand temporal relationships between FSH and GnRH, GnRH secretory patterns are overlaid (shaded patterns; scale not shown). Asterisks
identify statistically identified pulses of FSH. Arrows indicate the GnRH-associated bursts of FSH that occur on top of a previously triggered
episode of FSH release.

episodic burst of FSH in hypophyseal portal plasma. In con-
trast, such an association was not evident between GnRH
and jugular FSH. FSH remained elevated between episodes
of release, whether measured at the hypophyseal portal or
jugular levels. The interval between the onset of the GnRH
burst and secreted FSH in the hypophyseal portal blood was
very short, with initial increases occurring within the same
5-min sample for both hormones. The subsequent fall in
secreted FSH was rapid, but slower than that for GnRH.
Various characteristics of FSH pulses identified in the hy-
pophyseal portal and peripheral circulation are summarized
in Table 1. Shown in Table 1A for each ewe are the sampling
duration, number of portal and jugular FSH pulses identi-
fied, and number of pulses per h. Previously reported pulse
numbers of GnRH from the same ewes (14) are provided in
parenthesis for comparison. Within each series, although the
number of LH pulses identified in the hypophyseal portal
plasma was the same as the number identified for GnRH (12),
the number of FSH pulses identified in the hypophyseal
portal plasma far exceeded that for GnRH. The mean pulse
 FSH SECRETORY DYNAMICS 427

FIG. 3. Secreted (hypophyseal portal

less jugular) FSH and jugular FSH pat-
terns from one ovariectomized ewe (no.
6). To understand temporal relation-
ships between FSH and GnRH, previ-
ously reported GnRH secretory pat-
terns (14) are overlaid. FSH and GnRH
results are plotted on logarithmic scale

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to facilitate evaluation of the magni-
tude of changes across series. Note that
the dominant mode of FSH secretion is
basal, with episodes of FSH secretion
occurring on top of the basal secretion.
The thin line patterns on either side of
the plotted concentrations of secreted
FSH represent the range of duplicate
values. When the lines cannot be dis-
cerned, they are within the size of the
symbol.

FIG. 4. Secreted (hypophyseal portal

less jugular) FSH and jugular FSH pat-
terns from another ovariectomized ewe
(no. 10). For details, see Fig. 3.

amplitude of hypophyseal portal FSH is shown in Table 1B.
Portal FSH pulses were 18.0 6 5.5-fold greater in amplitude
than jugular FSH pulses. Further, GnRH-associated pulses of
FSH were larger (P , 0.01) than non-GnRH-associated pulses
of FSH (57.7 6 10.8 vs. 43.4 6 11.4 ng/ml, respectively; Table
1C). Such a relationship was not evident when the analysis
was conducted on jugular FSH pulses (3.6 6 0.8 vs. 3.2 6 0.3).
The disappearance time of FSH in the hypophyseal portal
plasma, although not as rapid as that reported for GnRH
(1.7 6 0.2 min), was much faster than that observed for FSH
in the periphery (7.8 6 2.3 vs. 26.2 6 5.5 min for hypophyseal
portal and peripheral FSH, respectively; Table 1B).
Table 2 summarizes average concentrations of FSH in the
hypophyseal portal and peripheral circulations during the
6-h collection period and the relative proportions of the basal
and episodic components. The total FSH level measured in
hypophyseal portal blood during the 6-h collection period
was approximately 21-fold greater than that measured in
peripheral blood (2919.8 6 1023.5 vs. 138.9 6 38.2 ng/ml,
respectively). Total FSH measured in the basal compartment
428 FSH SECRETORY DYNAMICS Endo • 1997
Vol 138 • No 1

TABLE 1. Characteristics of FSH pulses identified in hypophyseal portal and peripheral circulation
A. Number of FSH pulses identified in hypophyseal portal and jugular circulation

Animal No. of S-FSH No. of S-FSH No. of J-FSH No. of J-FSH

Hours
ID peaksa pulses/h peaks pulses/h
Ewe 1 6 10 (8) 1.7 9 1.5
Ewe 2 6 14 (9) 2.3 12 2.0
Ewe 3 12 18 (16) 1.5 25 2.1
Ewe 5 6 14 (8) 2.3 12 2.0
Ewe 6 6 12 (9) 2.0 10 1.7
Ewe 10 6 10 (8) 1.7 16 2.7
Mean 1.9 6 0.1 2.0 6 0.2

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(1.4 6 0.04)
a
Numbers in parentheses depict corresponding no. of GnRH pulses. S-FSH, secreted FSH; J-FSH, jugular FSH.
B. FSH pulse characteristics in hypophyseal portal and peripheral circulation

Amplitude (ng/ml) Half disappearance (min)

Animal ID
Portal Jugular Portal Jugular
Ewe 1 97.6 2.3 3.4 21.0
Ewe 2 45.2 2.9 3.6 9.6
Ewe 3 60.1 5.6 6.9 48.7
Ewe 5 26.5 2.2 6.6 23.3
Ewe 6 66.0 2.9 18.7 33.8
Ewe 10 19.4 4.5 7.8 20.6
Mean 6 SE 52.5 6 11.7 3.4 6 0.6 7.8 6 2.3 26.2 6 5.5
C. FSH pulse amplitudes: GnRH-associated vs. non-GnRH-associated

GnRH-Associated (ng/ml) Non-GnRH-associated (ng/ml)

Animal ID
Portal Jugular Portal Jugular
Ewe 1 100.3 2.2 87.5 2.7
Ewe 2 53.7 2.8 36.1 3.0
Ewe 3 62.3 7.1 45.0 3.7
Ewe 5 35.6 2.3 17.9 2.1
Ewe 6 68.7 2.7 61.1 3.0
Ewe 10 25.4 4.5 12.9 4.4
Mean 6 SE 57.7 6 10.8 3.6 6 0.8 43.4 6 11.4 3.2 6 0.3

of FSH secretion during the 6-h collection period averaged
2245.1 6 897.0 and 101.5 6 36.5 ng/ml in hypophyseal portal
and jugular circulations, respectively. Although basal GnRH
concentrations were reported to be at or near the limit of
detection (14), the mean baseline of hypophyseal portal FSH
was 8.2 6 2.4-fold greater than that estimated for peripheral
FSH (68.4 6 16.6 vs. 9.1 6 1.0 ng/ml, respectively). A sub-
stantially greater proportion of FSH comprised the basal
component compared with the episodic component (72.9 6
3.8% vs. 27.1 6 3.8%, respectively; P , 0.01). A similar re-
lationship was noted for jugular FSH (69.2 6 6.5% vs. 30.8 6
6.5%, respectively; P , 0.01).
Figure 5 summarizes the pulse concordance relationships
of GnRH and FSH (solid bars), with values for GnRH and LH
provided for comparison in the background (striped bars). As
reported earlier, a virtual one to one relationship exists be-
tween GnRH and LH, whether LH was measured at the
hypophyseal portal (12) or peripheral level (14). Similarly,
assessment of FSH in hypophyseal portal blood revealed a
close association between GnRH and hypophyseal portal
FSH, with 93% of the GnRH pulses found to be associated
with FSH pulses. This relationship was less pronounced
when GnRH and jugular FSH pulses were compared (79%
concordance). Episodes of FSH secretion were identified in
the absence of corresponding GnRH pulses (31.5% in hy-
pophyseal portal and 45% in peripheral blood).
The time lag relationship between the onset of GnRH and
hypophyseal portal FSH pulses closely paralleled that of
hypophyseal portal LH (1.2 6 0.3 min for LH and 1.1 6 0.4
min for FSH). No time lag existed between the onsets of LH
and FSH pulses (0.4 6 0.2 min).
Discussion
Progress in understanding the secretory nature of FSH has
been limited by the inability to assess secretory patterns of
FSH at a site close to its production. In the majority of studies
in which secretory patterns of FSH were assessed from the
peripheral circulation, the very nature of an episodic pattern
of FSH secretion appears suspect. As a consequence, many
studies that have characterized LH patterns in depth are
either limited by infrequent measurements of FSH or by the
use of mathematical procedures to deconvolve the nature of
secretion (9, 10, 19 –21). This difficulty can be overcome if
blood is acquired close to the site of secretion before the
secretory products are subjected to dispersion and clearance
in circulation. Recently, we determined that hypophyseal
portal blood, in addition to serving as a source material for
defining secretory patterns of hypothalamic secretions (11,
13), provides a means to define secretory patterns of pituitary
secretions (12). Exploiting this powerful approach we find, as
has been surmised (2– 6), that FSH is secreted in two modes:
 FSH SECRETORY DYNAMICS 429

TABLE 2. Average concentrations of FSH in the hypophyseal portal and peripheral circulations and relative proportions of basal and
episodic components
A. Total and basal components of FSH

Total (basal 1 pulsatile), ng/ml Total basal, ng/ml

Animal ID
Portal Jugular Portal Jugular
Ewe 1 7763.2 98.9 6540.1 81.6
Ewe 2 3449.3 321.5 2565.5 280.5
Ewe 3a 2089.6 121.8 1515.2 53.8
Ewe 5 1480.5 84.9 1071.3 64.4
Ewe 6 1606.4 63.8 903.1 40.5

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Ewe 10 1129.8 142.8 875.6 88.4
Mean 6 SE 2919.8 6 1023.5 138.9 6 38.2 2245.1 6 897.0 101.5 6 36.5
a
Total assessed for 12 h and divided by 2.
B. Proportion (%) of FSH in basal and pulsatile components

Basal component Pulsatile component

Animal ID
Portal Jugular Portal Jugular
Ewe 1 84.2 82.5 15.8 17.5
Ewe 2 74.4 87.2 25.6 12.8
Ewe 3 72.5 44.2 27.5 55.9
Ewe 5 72.4 75.9 27.6 24.0
Ewe 6 56.2 63.3 43.8 36.5
Ewe 10 77.5 61.9 22.5 38.1
Mean 6 SE 72.9 6 3.8 69.2 6 6.5 27.1 6 3.8 30.8 6 6.5

caveats, studies in rats have documented clearly discernible

FIG. 5. Pulse concordance relationships of hypophyseal portal FSH to
GnRH, peripheral FSH to hypophyseal portal FSH, and GnRH to
hypophyseal portal FSH. For comparison, corresponding relations of
LH are provided in the background (striped boxes).
tonic (basal secretion) and episodic. In addition, we find that
the episodic mode of secretion includes both GnRH-associ-
ated and non-GnRH-associated episodes of FSH secretion.
Whether the basal mode results from additional minor ep-
isodes of non-GnRH pulsing or continuous secretion remains
to be determined. Furthermore, the results of this study pro-
vide direct evidence that unlike LH, which is secreted pri-
marily in pulses, the predominant mode of FSH secretion is
basal.
Episodic mode of FSH secretion
Although part of the problem in assessing secretory dy-
namics of FSH in the periphery has stemmed from its long
half-life (22–24), it has also been confounded by the molec-
ular heterogeneity of FSH (25). FSH isoforms secreted in
pulses appear to have a much shorter half-life compared with
those secreted in between pulses (8, 26). Despite these
episodic patterns of circulating FSH (27). However, because
a-subunit is also secreted in a pulsatile manner (28), and a
cross-reactivity in the FSH assay was not assessed, these
studies did not discern whether the observed pulsatility re-
sulted primarily from episodic secretion of FSH or pulsatile
secretion of the a-subunit. Studies in sheep (7, 8) and humans
(10) have failed to show discrete pulses of FSH such as those
seen for LH and have relied heavily on statistical approaches
to deconvolve pulses (29). Our own studies in nutritionally
growth-retarded ovariectomized lambs, in which GnRH se-
cretion is dependent on the nutritional status of the animal,
have shown the existence of bioactive, but not clearly de-
finable (except by pulse analysis), immunoreactive pulses of
FSH (8). Supportive evidence for a GnRH-driven episodic
component of FSH has come from perifusion studies (30 –32)
in which FSH secretion mirrors the delivered pulsatile pat-
tern of GnRH.
Using cavernous sinus sampling as an approach to get
closer to the secretion site, Irvine and Alexander (33) char-
acterized the secretory pattern of FSH during the luteal phase
of the cycle in horses, a time when GnRH pulse frequency is
expected to be low. They identified eight pulses of concurrent
LH and FSH in both the pituitary and peripheral blood
during 80 h of sampling. Unfortunately, this study, similar
to those in rats, did not provide an estimate of the a-subunit
cross-reactivity of the FSH assay. Using a well characterized
FSH assay (15–17) that cross-reacts minimally with a-subunit
(,0.3%) and a unique approach for characterizing FSH se-
cretion dynamics in vivo, our studies extend the observations
from the mare and unequivocally demonstrate that an epi-
sodic component of FSH secretion exists.
Key to our understanding of whether other hypothalamic
factors regulate pulsatile control of FSH secretion is the de-
termination of whether all identified pulses of FSH are con-
430 FSH SECRETORY DYNAMICS
current with GnRH pulses. In contrast to studies in the mare
(33), in which 35% of the identified GnRH pulses had no
concurrent FSH or LH pulses, almost all (93%) of the GnRH
pulses in this study were associated with FSH pulses. Such
a close relationship was, however, not evident when FSH
pulses were identified in the peripheral circulation. The very
discrete nature of the GnRH-associated bursts of FSH in the
hypophyseal portal blood and the close time lag relationship
between GnRH and FSH suggest that the primary factor
responsible for induction of the GnRH-associated pulses of
FSH is GnRH.
Interestingly, secretory excursions of FSH were also iden-
tified in the absence of detectable GnRH pulses. Further-
more, many of the GnRH-associated pulses of FSH them-
selves appeared to develop on top of a previously elicited
episode of FSH release. In forming a judgment regarding non
GnRH-associated episodic FSH secretion, one should bear in
mind that the blood passing from the hypophyseal-portal
circulation is not returned; washout can lead to very fast
disappearance times. Thus, simple persistence of concentra-
tions is indicative of additional FSH release; the fact that the
concentrations increase provides evidence for incremental
active secretion. The existence of fairly discrete, non-GnRH-
associated excursions of FSH suggests external coordination
of the gland by some trigger, rather than independent ac-
tivity within the pituitary gland. An understanding of what
controls the non-GnRH-associated component of episodic
FSH secretion is critical to our understanding of the control
of the secretion of this hormone. Several possibilities are
plausible. First, the non-GnRH-associated excursions of FSH
could be the outcome of intrinsic pituitary FSH rhythmicity.
This appears unlikely, because long term perifusion studies
of dispersed ovine pituitary cells fail to show such an epi-
sodic pattern of secretion (our unpublished data). Second,
non-GnRH-associated episodes of FSH secretion could rep-
resent FSH responses to low levels of GnRH (not detectable
by RIA and subthreshold for LH). This also seems unlikely
because dose-response studies carried out in static cultures
(our unpublished data) and in nutritionally growth-
restricted ovariectomized lambs (34) show FSH not to be
more sensitive to GnRH. Furthermore, preliminary studies
that used GnRH antagonists to block GnRH input in ovari-
ectomized ewes show that such excursions in FSH persist
even after complete blockade of GnRH action (35). Another
possible explanation is that non-GnRH-associated release of
FSH is caused by acute changes in locally produced inhibin,
activin, and/or follistatin (36 – 40). This, however, seems un-
likely in view of the findings that these FSH regulatory pep-
tides take long to act and have a sustained effect on the basal
FSH secretion (41– 43), as opposed to the rather discrete FSH
secretory episodes that were often observed in this study.
Another possibility is that the non-GnRH-associated epi-
sodes of FSH release are the outcome of inputs from a se-
lective FSH-releasing-factor(s) originating from the hypo-
thalamus. Studies demonstrating selective regulation of FSH
release after ablation (44), deafferentation (45), or destruction
(46) of the dorsal anterior hypothalamic area or electrochem-
ical stimulation of hypothalamic regions apart from those
that regulate LH secretion (47) strongly support a specific site
of control for FSH release. Evidence supporting the existence
Endo • 1997
Vol 138 • No 1
of FSH-releasing factor was first provided by Igarashi and
McCann (48). Although considerable anatomical and bio-
chemical evidence supports this possibility (49), and partial
separation of a separate FSH-releasing activity has been
achieved (50), no specific FSH-releasing factor has been iso-
lated or found to be released into hypophyseal portal blood.
Whether the non-GnRH-associated excursions in FSH secre-
tion represent responses to a yet to be identified hypotha-
lamic FSH-releasing factor remains to be determined.
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Basal component of FSH secretion
In addition to the episodic component of FSH release dis-
cussed, several lines of evidence have suggested that a large
portion of circulating FSH results from basal secretion. First,
circulating FSH concentrations remain detectable for several
days in sheep after interruption of hypothalamic inputs to
the pituitary (51) and in hypophysectomized rats bearing
pituitary transplants under the kidney capsule (52). Second,
FSH continues to be secreted in long term pituitary cultures
(53) while LH secretion declines. The in vivo studies dis-
cussed earlier (7–10, 51, 52), have used peripheral FSH mea-
surements and do not provide direct evidence for a basal
mode of FSH secretion. The results of this study provide
direct evidence that not only does a basal mode of FSH
secretion exist, but this is the dominant mode of FSH secre-
tion in ovariectomized ewes.
Considering that the dominant component of FSH secre-
tion is not episodic in the employed ovariectomized model
with its high frequency, high amplitude patterns of GnRH
(11, 54), the relevance of the pulsatile component of FSH
release during the estrous cycle needs to be addressed. Here,
GnRH pulses are of much lower amplitude (54). GnRH neu-
tralization studies have revealed that blockade of GnRH
input, while having little effect on peripheral FSH secretion
(55), may be relevant in inducing paracrine factors, such as
follistatin (56, 57), that may be involved in the control of basal
FSH secretion.
Hypophyseal portal blood as a means to assess active
secretion of FSH
Demonstration of the two modes of FSH secretion was
made possible because the secretory signal can be monitored
with high resolution in hypophyseal portal blood free from
the influence of dispersion and clearance in the circulation.
Because rates of changes in hormone concentrations in hy-
pophyseal portal samples are far more rapid than those in the
periphery, we believe that FSH patterns in the hypophyseal
portal samples approximate the actual secretory dynamics of
FSH more closely than those obtained by other reported
approaches.
An important caveat that needs to be addressed relates to
the means by which FSH enters the hypophyseal portal cir-
culation. Does FSH in hypophyseal portal blood represent
leaching from damaged cells or active secretion? The dis-
creteness of the LH and FSH episodes, the one to one rela-
tionship of GnRH with LH and FSH, their immediate block-
ade after GnRH antagonist administration (35), and the
constancy of secretion (perifusion studies suggest that dam-
aged cells deplete their content and do not respond to secre-
 FSH SECRETORY DYNAMICS
tagogues) all suggest that the FSH we measure in pituitary
portal blood reflects primarily secretion and not leakage
from damaged pituitary cells. As for the site of origin of the
FSH in hypophyseal portal blood, there are three possibili-
ties: 1) active secretion of gonadotropes located in pars tu-
beralis, 2) retrograde blood flow from pituitary to the hy-
pothalamus, and 3) drainage from pituitary sinusoids.
Secretion vs. clearance
Considering that hypophyseal portal measurements pro-
vide more accurate assessment of what is being secreted, an
important question arises. How meaningful are jugular FSH
measurements in assessing FSH secretory dynamics? This
question is particularly relevant because peripheral blood is
the only convenient means available for monitoring FSH.
Because peripheral measurements are influenced by the rates
of secretion, clearance, and degradation, caution needs to be
exercised when drawing conclusions concerning the secre-
tory nature of FSH from peripheral measurements. Clearly,
some conclusions, such as the dual mode of FSH release, hold
true regardless of whether the measurements are made at the
hypophyseal portal or peripheral level. Nonetheless, periph-
eral measurements have failed to provide a true nature of
secretory events such as the discreteness of the GnRH-asso-
ciated pulses of FSH and the close time lag relationships of
GnRH and FSH.
As a result of dispersion, the fold difference in hypo-
physeal portal and peripheral FSH concentrations was much
higher when the comparison was made at the pulsatile com-
ponent of release (z18-fold) than at the basal component of
release (z8-fold). Additionally, the half-time disappearance
rate of FSH pulses at the periphery, about 25 min, is shorter
than the reported half-life of FSH (3– 6 h) (22–24). These
results suggest that FSH secreted in pulses may be cleared
faster from the circulation than that secreted in the tonic
mode. Because assessments of half-life in the past have been
computed on the basis of exogenous FSH, the long half-life
estimates computed this way may reflect the nature of ad-
ministered FSH and may not apply to what is secreted within
a FSH pulse. Supportive evidence for such a concept comes
from our studies in patients with Kallman’s syndrome in
whom administration of GnRH led to the release of short-
lived isoforms of FSH (26).
The difference in half-life, however, does not explain why
the relative magnitude of changes in LH and FSH differed
within a given sample between the hypophyseal portal and
peripheral circulations. These differences, although intrigu-
ing, lend support to the view that gonadotropes are distrib-
uted differentially among different portions of the pituitary
(58, 59). Gonadotropes are comprised of a heterogeneous
population of cells, some producing LH, some producing
FSH, and others producing both (60). Therefore, from a quan-
titative perspective, the proportions of LH and FSH mea-
sured in the hypophyseal portal blood circulation appear
more a representation of their production in the lesioned part
of the pituitary and not a quantitative estimate of pituitary
secretion as a whole. Because the sampled hypophyseal por-
tal plasma may represent an unknown fraction of the total
secretory output, the patterns of FSH in the hypophyseal
431
portal plasma should be used merely as guides, providing an
insight into the dynamics of pituitary FSH secretion, and not
to calculate total secretion.
In summary, characterization of FSH secretory profiles in
the hypophyseal portal blood from ovariectomized ewes
reveals a dual mode of FSH secretion, basal and episodic.
Furthermore, a close one to one relationship exists between
GnRH and FSH pulses, suggesting that GnRH is a regulator
of episodic FSH secretion. Finally, identification of non-
GnRH-associated excursions of FSH secretion supports the
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possibility that other, yet to be identified, factors may be
involved.
Acknowledgments
The authors acknowledge the efforts of Geoffrey E. Dahl, Neil P.
Evans, Douglas L. Foster, Judy M. Manning, and Sue M. Moenter in
collecting the samples analyzed in this study.
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