| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Station de Physiologie de la Reproduction des Mammifères Domestiques, Institut National de la Recherche Agronomique (A.C.), 37380 Nouzilly, France; and Reproductive Sciences Program, University of Michigan (F.J.K.), Ann Arbor, Michigan 48109; and the Laboratory of Neuroendocrinology, The Babraham Institute (A.H.), Cambridge, United Kingdom CB2 4AT
Address all correspondence and requests for reprints to: Dr. A. Caraty, Station de Physiologie de la Reproduction des Mammifères Domestiques, Institut National de la Recherche Agronomique, 37380 Nouzilly, France.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-immunoreactive cells of the hypothalamus
revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants
located bilaterally within or adjacent to the area of estrogen
receptor-expressing cells of the ventromedial nucleus. Two of these
ewes exhibited a LH surge without showing any form of estrogen negative
feedback. In the second experiment, we used the technique of
hypophyseal portal blood collection to monitor GnRH secretion directly
at the time of the LH surge induced by estradiol delivered either
centrally or peripherally. Central estradiol implants induced the GnRH
surge. The duration and mean plasma concentration of GnRH during the
surge were not different between animals given peripheral or central
MBH estradiol implants. Cholesterol-filled MBH microimplants did not
evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the rat, there is good evidence that estrogen acts within the medial preoptic area (MPOA) to activate GnRH neurons. The insertion of estradiol microimplants into the MPOA, but not the mediobasal hypothalamus (MBH), evokes a LH surge in ovariectomized rats (14) and microimplants of ER antagonists into the MPOA abolish the LH surge (15). Although the distribution of ER-containing cells has now been identified in the ovine hypothalamus (12, 16, 17), the estrogen-sensitive brain regions required for the positive feedback effects of estradiol on GnRH secretion in the ewe have not been identified. The first study to use local implantation of estradiol into the brain of the ewe reported on only two animals and provided inconclusive evidence regarding the site(s) of estrogen action for stimulating LH secretion (18). A more recent report found that the placement of estradiol microimplants into the MBH, but not the anterior hypothalamus or POA, elicited both estrous behavior and LH secretion in breeding season ewes (19). However, the LH surges induced by central estradiol in that study occurred in only 25% of animals and were small in nature. Given that only a small fraction of the total GnRH surge is needed to induce a full-sized LH surge in the ewe (20), full amplitude GnRH surges may not have been induced in that study by the brain estradiol microimplants. Further, as estradiol can stimulate the pituitary directly in the ewe (21), a positive action of estradiol at the pituitary in the presence of a constant regimen of GnRH release could not be totally excluded (22, 23). In addition, as estrous behavior was monitored repeatedly in these experiments after estradiol administration, gonadotropin secretion may have been stimulated by the presence of the ram (24). Thus, it is important to determine the effect of estradiol microimplants on GnRH secretion itself. Failure to elicit a normal GnRH surge would suggest that other neural sites contribute to the preovulatory GnRH surge.
In the present series of studies, we have examined where estradiol acts within the hypothalamus of the ewe to elicit LH and GnRH surges. In the first part of the study, we used the stereotaxic placement of estradiol microimplants in the MPOA and MBH to examine the local actions of estrogen in these structures with reference to both its negative and positive feedback effects on LH secretion. In the analysis of this study, we paid particular attention to the proximity of the bilateral estradiol microimplants to the ER-expressing cell groups of the hypothalamus. In the second part of the study, portal blood was sampled to determine whether the positive feedback actions of central estradiol microimplants were attributable to stimulation of GnRH secretion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cerebral implant surgery
Each ewe was anesthetized (Halothane, Pitman-Moore France,
Meaux, France), and its head was positioned in a stereotaxic frame
(Précision Cinématographique, Paris, France). One
milliliter of radioopaque liquid (Omnipaque, Nycomed Ingenon, Paris,
France) was injected into the lateral ventricle, and frontal and
lateral x-rays were taken to give specific landmarks of the ventricular
system. The massa thalamica as well as the infundibular recess were
used as internal landmarks for the antero-posterior and vertical
orientations, and the middle of the third ventricle was used as a
landmark for laterality. Stainless steel guide cannulas (od, 1.20 mm;
id, 0.80 mm) were implanted to a depth 5 mm above the target site. Ewes
received bilateral guide tube implants in the MPOA (Exp 1) or MBH (Exp
1 and 2). The stereotaxic placement of the MPOA implants was 32 mm
anterior to the interaural line (anterior-posterior = 32), 1.0 mm
lateral to the midsagittal plane (lateral = 1.0), and within 0.5
mm of the base of the third ventricle (dorsal = 0.5). Two
stereotaxic placements were used for the MBH: one for the lateral
division of the ventromedial nucleus (VMN; anterior-posterior = 28;
lateral = 2.5; dorsal = 1.5) and one for the arcuate nucleus
(ARN; anterior-posterior = 28; lateral = 1; dorsal = 0.5).
Dental acrylic cement was applied to hold the guide tubes rigidly in
position, and the tubes were plugged. The whole device was protected by
a threaded cylindrical Teflon cap (od, 30 mm; id, 22 mm; height, 22
mm), which was anchored to the skull with four stainless steel screws
and dental acrylic cement. After surgery, ewes were injected daily with
antibiotics (5 ml Chloram-Frecortyl, Vetoquinol, Lure, France) for 5
days and diuretics (3 ml Diurizone, Vetoquinol) for 3 days.
The microimplants were made of stainless steel tubing (od, 0.70 mm; id, 0.45 mm) and filled with either crystalline estradiol (17ß-estradiol, Sigma Chemical Co., L’Isle D’Abeau Chesnes, France) or cholesterol by tamping the tip of the tube into pure molten substance. After crystallization, the outer surface of the tube was carefully cleaned with ethanol to remove any steroid residue. The presence of steroid in the lumen of the tube was verified under a dissecting microscope. Before use, the implants were subjected to formol vapors for 12 h to ensure sterility.
Experimental design
Exp 1: effect of brain estradiol implants on LH secretion.
The objective of this experiment was to determine whether local
estradiol administration into the MPOA or the MBH could induce a LH
surge. However, special attention was also given to a possible initial
negative feedback effect of estradiol as an additional marker of
estradiol efficacy in each site. Twenty Ile de France ewes exhibiting
regular estrous cycles were used during the breeding season. Seven
animals received bilateral guide cannulas directed at the MPOA and 13
at the MBH. For the latter, guide tubes were positioned so as to
provide microimplants that would involve either the VMN or the ARN, the
two ER-rich nuclei of the MBH (16, 17).
One month later, ewes were ovariectomized and run through an artificial estrous cycle by manipulation of peripheral estradiol and progesterone implants. On the day of ovariectomy, animals were treated immediately with an intravaginal controlled internal drug device (CIDR) progesterone device (InterAG, Hamilton, New Zealand) and a sc 10-mm SILASTIC brand implant (Dow Corning, Midland, MI) containing estradiol to simulate the midluteal phase of the estrous cycle (28). Luteolysis was simulated 12 days later by removal of the CIDR. Sixteen hours later, central estradiol microimplants were inserted in the cerebral guide tubes. Jugular blood samples were taken every 10 min for 6 h during two periods: the first before insertion of the brain estradiol implants (i.e. between 10–16 h after progesterone removal) and the second between 4–10 h after microimplant insertion, during the anticipated negative feedback response. Blood samples were then taken every 2 h for the following 24-h period during the anticipated LH surge.
The next day, peripheral (10-mm; sc) and brain estradiol implants were removed, and a CIDR was inserted to elevate progesterone, a treatment reported to increase the number of ER-expressing cells (17). Ten days later, animals were killed and decapitated, and both carotid arteries were catheterized. Two liters of physiological saline containing 1% sodium nitrite followed by 4 liters of cold modified Zamboni’s fixative (17) were perfused using a pump over a period of about 15 min. The brains were then removed, and a block of tissue containing the POA and hypothalamus was dissected and placed in the fixative solution for a further 24 h. The tissue block was subsequently immersed in a 15% sucrose, Tris-buffered saline (TBS) solution at 4 C. Two to 3 days later, 60-µm thick coronal sections though the POA and hypothalamus were cut on a freezing microtome and stored in cryoprotectant (29) at -20 C until histological and immunocytochemical analyses were performed.
Exp 2: effect of estradiol implants on GnRH secretion. As the first experiment revealed that estradiol implants in the MBH evoked a LH surge, the objective of this experiment was to determine whether these LH surges were a consequence of a GnRH surge into the hypophyseal portal blood. The study was conducted during the breeding season, using 30 ewes over a period of 3 yr. Animals exhibiting regular estrous behavior were implanted bilaterally with guide cannula directed at the MBH. One month later, they were ovariectomized and run through two sequential artificial estrous cycles. The first cycle, undertaken as described above, was used to determine which animals treated with brain estradiol implants exhibited a LH surge. During the artificial follicular phase, jugular blood was sampled hourly by venipuncture from 10–35 h after insertion of the central estradiol implants. The next day, the estradiol implants were removed, a CIDR progesterone device was inserted, and the animals were run through a second artificial cycle.
During the luteal phase of this second cycle, ewes were anesthetized and fitted with an apparatus for the collection of pituitary portal blood as described previously (25, 26). The LH secretion profile of the first cycle was used to allocated animals to three different experimental groups. Animals showing a LH surge (definition below) were allocated either to the experimental group receiving bilateral brain estradiol implants for the second time (n = 13) or to the negative control group receiving central cholesterol implants (n = 5). In both cases, the brain implants were inserted 16 h after withdrawing progesterone. Animals not exhibiting a LH surge in the first cycle were used for the positive control group (n = 10). These ewes were treated with four sc 30-mm SILASTIC estradiol implants, sufficient to rise circulating estradiol concentrations to a peak follicular phase level (26), in addition to central cholesterol implants. Portal and jugular blood were collected hourly between 10–40 h after the implantation of estradiol or cholesterol. Preliminary experiments had demonstrated that animals exhibiting a LH surge after brain estradiol implantation in the MBH showed another LH surge in a second successive artificial cycle using estradiol implants in the same location (five of five animals; our unpublished results).
Immunohistochemistry
Immunocytochemistry for the ER was carried out on free-floating
sections using the ID5 monoclonal mouse antibody generated against the
N-terminal region of the human ER (30), as described previously (31).
Before immunostaining, sections were washed in 40% methanol-TBS, pH
7.4, containing 1% H2O2 for 10 min followed by
two washes with TBS. Sections were then incubated with the ID5 antibody
diluted 1:10 in TBS containing 2% normal horse serum, 1% BSA, and
0.3% Triton-X for 40 h at 4 C. After additional washes in TBS,
sections were incubated in biotinylated horse antimouse Igs (1:300;
Vector Laboratories, Burlingame, CA) for 90 min at room temperature
followed by avidin-peroxidase complex (1:50; Vector Elite kit, Vector),
also for 90 min at room temperature. A glucose
oxidase-nickel-diaminobenzidene technique was used to visualize the ER
immunoreactivity (11, 13, 31). Sections were mounted on gelatinized
slides and viewed on a DM-RB Leitz microscope (Leitz, Rockleigh,
NJ).
RIAs
Concentrations of GnRH in portal plasma (Exp 2) were measured by
RIA in duplicate aliquots after methanol extraction using a specific
antibody against the C-terminal portion of the molecule as described
previously (32). GnRH assay sensitivity averaged 0.2 pg/tube (10
assays), and the intraassay coefficient of variation averaged 13%. LH
was measured (Exp 1 and 2) in duplicate aliquots of plasma by a
specific RIA (33). The intrasensitivity of the assay was 0.1 ng/ml
standard CY1051 (0.25 ng/ml NIH LH-S1). The intra- and interassay
coefficients of variation were 8% and 12%, respectively.
Data analysis
Exp 1. The negative feedback action of estrogen on LH
secretion was determined by comparing the mean plasma LH concentration
6 h before and after estradiol implantation.
Animals were classified into three groups; 0 = no effect on LH
secretion (change does not exceed 20% of the pretreatment level, 
4
times theSD of the mean level); 1 = moderate degree of
negative feedback (mean LH after estrogen treatment is reduced by
20–50%); and ** = strong negative feedback (suppression of mean LH
after estrogen treatment exceeds 50%). Comparison of mean plasma LH
concentration before and after estradiol was performed using Student’s
paired t test.
For the positive feedback effects of estrogen, the individual profiles of LH secretion were used to classify animals as exhibiting no surge (score = 0; LH levels did not rise above 2 times the preestrogen concentration), a small surge (score = 1; when LH values exceeded 10 ng/ml but were less than 20 ng/ml for at least two consecutive samples and were twice the preestradiol concentration), or a large amplitude LH surge (score = **; when values increase above 20 ng/ml for at least two consecutive samples and were twice the preestradiol concentration).
The exact position of each estradiol implant within the MPOA or MBH was determined in counterstained histological sections, and their relationship to ER-expressing cells was determined in sections immunoreacted for the ER. Each implant was scored in relation to its position to the ER-immunoreactive cells comprising individual hypothalamic nuclei (0 = not adjacent, i.e. >200 µM from ER-expressing cells, 1 = adjacent to and touching ER-expressing cells; 2 = embedded within the ER-expressing cell group).
Exp 2. For the first cycle, occurrence of the LH surge was defined as described above. For the second cycle the definition of the surge was different because the portal collection reduced LH surge amplitude, and it was necessary to describe surge characteristics (onset, duration, and amplitude). For the second cycle, onset of the GnRH or LH surge was defined as the time at which LH or GnRH concentrations exceeded the presurge baseline by 3 SD and remained elevated for at least 2 h. The end of the LH surge was defined as the time LH concentrations fell below 3 SD above the presurge baseline. The end of the GnRH surge was more difficult to identify due to its protracted phase of decline. Thus, the end of the GnRH surge was defined as the time GnRH levels had fallen 80% from the surge maximum level (defined as the mean of the three highest GnRH values during the surge). The mean plasma concentration during the surge was calculated for each hormone by summing the concentrations during the surge and dividing this sum by the duration. The onset of the LH and GnRH surge, duration, and mean plasma concentrations of LH and GnRH during the surge were compared between animals receiving estradiol implants peripherally or centrally using Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The remaining ewe
had one of its implants located within the rostral third ventricle and
was omitted from the analysis. The implants of individual animals were
spread throughout the MPOA and extended from the rostral POA at the
level of the organum vasculosum of the lamina terminalis (Fig. 1, ewe 18), to the caudal POA, and, in
the case of one ewe, the anterior hypothalamus (Table 1). All six ewes
exhibited a strong negative feedback response to estradiol
implantation, which resulted in a significant decrease in the mean LH
concentration (1.78 ± 0.25 vs. 0.63 ± 0.14
ng/ml; P < 0.001). Levels of LH returned to initial
preestradiol concentrations 12–15 h after estradiol implantation (Fig. 2, insets for ewes 2 and 18).
None of the six animals displayed any form of positive feedback of
estrogen on LH secretion (Table 1).
|
|
|
and 3 and Table 1). One additional
ewe had implants located above the MBH in the posterior hypothalamus
and dorsomedial nucleus (ewe 3), whereas the remaining two animals had
one implant located in the third ventricle and were omitted from the
analysis. Six of 11 of these animals with administration of estradiol
in the MBH showed negative feedback (Table 1). However, when analyzed
with respect to the depth of the implants within the MBH, it became
apparent that full or moderate levels of negative feedback were only
found when both implants were located in the ventral half of the MBH
(i.e. the middle or caudal VMN or ARN; as shown in Fig. 1
and Table 1). Thus, implants located in the mid- to caudal VMN of ewe
22 (Fig. 1) abolished LH pulsatility (Fig. 2) as did the bilateral ARN
implants (ewe 21, Fig. 2).
|
; animals 4, 13, 22, and 21).
However, in terms of estradiol positive feedback, the most striking
observation was that all 3 ewes (no. 10, 13, and 22) with both implants
located adjacent to or within the area of ER-expressing cells of the
VMN (Figs. 1 and 3) exhibited a
full-amplitude LH surge (Fig. 2). When extended to all ewes with
bilateral VMN implants, regardless of whether they were adjacent to the
ER-immunoreactive cells, 4 of 5 ewes exhibited a LH surge (Table 1).
The LH surge was less evident in ewes with at least one implant in the
ARN (Table 1.); only 1 of 4 such ewes displayed a normal LH surge,
whereas an additional animal, which had bilateral ARN implants (ewe
21), exhibited only a marked increase in LH secretion (Fig. 2). The ewe
with both implants outside the MBH (ewe 3) also exhibited only a marked
increase in LH secretion. In all cases, the onset of the LH surge
occurred between 16–28 h after brain estradiol implantation.
Interestingly, two of the VMN implant ewes (no. 4 and 13) exhibited a
full-amplitude LH surge without showing any form of negative feedback
(Fig. 2). For the 4 of 11 animals showing no increase in LH secretion,
LH concentrations returned to preestradiol levels within the 24-h
sampling period as did the POA-implanted ewes (data not shown). None of the three ewes with one implant in the third ventricle (one MPOA and two VMN implanted ewes) showed any form of positive feedback.
Exp 2: effect of estradiol implants on GnRH secretion
Of the 30 animals with bilateral MBH implants, 18 exhibited a
normal LH surge after intracranial estradiol implantation in the first
artificial estrous cycle. These 18 animals were allocated to either the
negative control group (MBH cholesterol implants; n = 5) or the
experimental group (MBH estradiol implants; n = 13) for the second
estrous cycle. Of the remaining 12 ewes, 10 animals showed no surge and
were used for the positive control group (sc estradiol implants plus
MBH cholesterol implants). The 2 remaining animals had only a small LH
surge and were excluded from the experiment.
Ten of the 13 animals receiving MBH estradiol during the second cycle
exhibited a surge of GnRH in portal blood coincident with the LH surge
in the peripheral circulation (Fig. 4, A
and B). Eight of the 10 animals in the positive control group treated
with peripheral estradiol and intracranial cholesterol implants also
exhibited simultaneous GnRH and LH surges (Fig. 4, C and D). Overall,
the pattern of GnRH secretion in these two groups was similar, although
there was considerable variation in the GnRH surge amplitude among
animals in both groups (Figs. 4 and 5). No significant differences were
found in the duration or mean plasma concentration of hormone release
during the GnRH and LH surges, but onsets of GnRH and LH surges were
earlier in animals treated with estradiol centrally compared with
peripherally (P < 0.05 for both). Postmortem
examination of the brains of the 5 animals in these two groups that
failed to show a LH surge (3 MBH estradiol, 2 peripheral estradiol)
revealed evidence of a large hemorrhage around the tip of 1 or both of
the estradiol/cholesterol implants. None of the 5 negative control
animals that received cholesterol implants in the MBH responded with a
GnRH or LH surge (Fig. 5, E and F).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Estrogen implanted into the immediate vicinity of the ventrolateral VMN induced the GnRH and LH surges. The lack of response to cholesterol implants indicated that this GnRH surge was not induced by mechanical stimulation of the VMN. Further, the characteristics of the GnRH surge in ewes with MBH estradiol implants were similar to those induced by peripherally administered estradiol. The large variation in GnRH surge amplitude in both peripherally and MBH-administered estradiol ewes is thought to be due to the position and extent of the portal vasculature that was cut for each individual portal blood collection (24). Of interest, the GnRH surges induced by central estradiol administration began slightly earlier than those induced by peripheral estradiol administration. This may reflect an earlier diffusion of the steroid into relevant brain centers as a first step of the positive feedback response. Alternatively, as the latency depends on the dose of estrogen (34, 35, 36), the early onset of the GnRH surge may indicate that relatively high concentrations of estradiol were achieved in the vicinity of the brain implant. Together, these observations indicate that neurons in the vicinity of and within the VMN are an important target for circulating estrogens in generating the normal GnRH surge.
The present findings provide an important extension of the studies reported by Blache and colleagues (19), who found that bilateral MBH estradiol implants generated a LH surge in 25% of ewes exposed to rams. Our present observations measuring GnRH in the absence of rams show that the MBH, and more specifically the vicinity of the ventrolateral VMN, is critical in the activation of GnRH secretion at the time of the normal estrogen-induced LH surge. Similar to that study (19), we found that the implants were most effective in generating the surge when positioned bilaterally in the vicinity of the VMN; unilateral MBH estradiol implants were ineffective in inducing an increase in LH secretion. Furthermore, ewes in which one of the implants was located in the third ventricle did not exhibit a LH surge. These results suggest that it is the bilateral activation of a localized group of VMN neurons that is required and that the general diffusion of estradiol within the ventricular system is not sufficient to generate the LH surge.
Bilateral estradiol implants within the MPOA were clearly ineffective as a site of estrogen positive feedback. We note that our implants in five individual ewes covered the entire rostro-caudal extent of the MPOA and, in one animal, were located within the anterior hypothalamic area. As none of these animals exhibited a LH surge, and the density of ER cells in the MPOA is actually greater than that in the VMN (31), it seems unlikely that estradiol diffusing from our implants might have missed some critical subpopulation of surge-specific estrogen-receptive neurons. This result is especially interesting as the antero-ventral periventricular nucleus within the rostral MPOA of the rat is believed to be the most important target for estradiol in the stimulating the LH surge (15, 37). In the present study, three of our ewes had bilateral implants in the rostral part of the MPOA, but no LH surge was induced. We do note, however, that the surges induced by implants in the VMN were obtained with a background of constant low concentrations of circulating estradiol. Hence, it is conceivable that low level estrogen exposure at one or several brain regions is required in a permissive manner for the rising follicular phase levels of estrogen to act within the VMN to evoke the GnRH surge.
Our conclusion that the VMN is an important site for estradiol positive
feedback in the ewe is strengthened by previous electrophysiological
and neuroanatomical observations. For example, approximately one third
of neurons located in the ovine MBH have been reported to increase
their level of electrical activity after iv estradiol administration
(38). Further, a large population of ER-containing cells has been
identified in the ventrolateral VMN of the ewe (16, 17), and a change
in the density of ER-immunoreactive cells has been found to occur
specifically within this region during the course of the estrous cycle
of the ewe (17). However, it must be pointed out that until now,
mapping of the distribution of ER cells by immunocytochemistry has been
limited to the -form of the ER. A recent report (39) examining the
distribution of ERß messenger RNA in the rat brain has suggested that
a low level of expression exists in the ARN and VMN. Although no data
on ERß expression are currently available in the sheep, it remains
possible that estradiol from our VMN implants may be activating one or
both types of ER.
The neurochemical identity and projections of ER-expressing cells of
the VMN of the ewe also are not well defined. As most GnRH neurons in
the ewe are located in the POA, the positive feedback signal is most
likely relayed from the VMN to a distal site. A recent study has
reported that a number of neurons in the VMN project to the ARN, but
few to the median eminence, of the ewe (40), and that a subpopulation
of VMN neurons also project to the MPOA (41). Our recent retrograde
labeling studies have shown that a large number of the VMN neurons
projecting to the vicinity of the GnRH cell bodies express ER
immunoreactivity (Herbison, A., and A. Caraty, unpublished). Hence, it
would appear that there are at least two routes by which estrogen,
acting within the VMN, may influence GnRH neurons in the ewe: one a
potentially direct input to preoptic GnRH cell bodies, and the other an
indirect pathway via neurons of the ARN. The neurochemical identity of
ER-expressing cells within the ovine VMN is not well characterized,
although preliminary observations have indicated that up to 70% of the
ER- immunoreactive cells in the ventrolateral VMN of anestrous ewes
synthesize somatostatin (42). Studies aimed at understanding the
neurochemical identity and projections of estrogen-sensitive VMN
neurons in the ewe are currently in progress.
In addition to determining where in the brain estradiol acts to induce a positive feedback on LH secretion, our studies also show that estradiol implanted in the MPOA or caudal MBH produces negative feedback. However, the interpretation of these results must be tempered by several questions related to our experimental approach. First, did estradiol produce negative feedback by diffusion to the pituitary gland? This seems highly unlikely in the case of the POA implants. The diffusion of radioactive estradiol from the type of implant used in this study is approximately 1 mm (19), and the POA of the ewe is located 8–10 mm from the pituitary. Further, estrogen implants located in the dorsal VMN, which is substantially closer to the pituitary than is the POA, exhibited no negative feedback. It remains possible, however, that the suppression of LH in response to estradiol implants in the ARN was due in part to diffusion to the pituitary gland. Second, was the dose of estradiol physiological? Using the same follicular phase model as that employed in the present study, Evans and colleagues (43) demonstrated that peripheral administration of physiological amounts of estradiol exerts a dose-dependent suppression of GnRH pulse size and a stimulation of pulse frequency during the breeding season of the ewe. This is different from the effect of estradiol observed in our study, in which visual examination of the pattern of secretion shows that the negative feedback action of estradiol results from either a slowing of LH pulse frequency or a complete abolition of pulsatile LH release. The results of the present study possibly reflect a response to a supraphysiological concentration of estradiol at the tip of the microimplants, as a large dose of estradiol given peripherally can induce a decrease in GnRH pulse frequency during the breeding season (44).
Despite these limitations, our findings raise the possibility that
estrogen-sensitive cells in both POA and MBH are capable of inhibiting
GnRH secretion in the ewe. MPOA implants inhibited LH regardless of
whether they were located in the rostral or caudal aspects of the
ER-expressing cells of this region. This suggests the neurons mediating
this inhibitory action are located either throughout or, allowing for a
1-mm radius of diffusion, centrally within the MPOA. The neurochemical
identity of the preoptic cells involved in this response awaits further
investigation. It is noteworthy that, as in the rat, many ER-expressing
cells in the ovine MPOA synthesize 
-aminobutyric acid (GABA) (13).
An estrogen-induced rise in preoptic GABA concentrations in the rat
(45) has been implicated in the inhibitory action of estrogen on LH
secretion within the MPOA of that species (46). Similarly, GABA has
been implicated in the suppression of LH before the surge in the ewe
(47). Regarding the MBH, only microimplants located in the ventral part
of this region show a clear negative feedback effect. As
ß-endorphin-, tyrosine hydroxylase-, and neuropeptide Y-containing
cells are present in this region (48) and possess ER (12, 31), they
are potential candidates for mediating the inhibitory effect of the
steroid on GnRH secretion.
Finally, an interesting and unanticipated result was obtained in Exp 1. Specifically, two ewes with VMN implants were found to exhibit a full LH surge without showing any evidence of estrogen-induced negative feedback. This unexpected observation raises the question of whether the positive and negative modes of estrogen feedback may be independent and mediated by different neuronal cell populations. An independence of the negative and positive feedback pathways has been found in MPOA-grafted hypogonadal mice, in which the ability to exhibit positive feedback was separated from the negative feedback response (49). Also consistent with this view is recent evidence suggesting the independent development of tonic and surge LH release mechanisms in the sheep during the course of sexual maturation (50). Clearly, additional animals and alternative experimental approaches are needed to determine whether estrogen negative feedback is a prerequisite for estrogen to induce positive feedback.
In summary our results show that placement of estradiol into the MBH, but not the POA, is able to induce a GnRH surge in the hypophyseal portal blood of the ewe. Although our findings do not exclude an action of estradiol elsewhere in the brain in enabling the GnRH surge to occur, they provide evidence that the VMN is an important site of action for the positive feedback signal in the ewe: the rise in circulating estradiol concentrations found during the follicular phase. Moreover, they also suggest that different sites mediate the positive and negative feedback actions of estradiol and that the LH surge can be induced independent of a prior negative feedback effect.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Lister Institute-Jenner Fellow.
Received September 25, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  R. L. Goodman, M. N. Lehman, J. T. Smith, L. M. Coolen, C. V. R. de Oliveira, M. R. Jafarzadehshirazi, A. Pereira, J. Iqbal, A. Caraty, P. Ciofi, et al. Kisspeptin Neurons in the Arcuate Nucleus of the Ewe Express Both Dynorphin A and Neurokinin B Endocrinology, December 1, 2007; 148(12): 5752 - 5760. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Roselli, H. Stadelman, R. Reeve, C. V. Bishop, and F. Stormshak The Ovine Sexually Dimorphic Nucleus of the Medial Preoptic Area Is Organized Prenatally by Testosterone Endocrinology, September 1, 2007; 148(9): 4450 - 4457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Arreguin-Arevalo, T. L. Davis, and T. M. Nett Differential Modulation of Gonadotropin Secretion by Selective Estrogen Receptor 1 and Estrogen Receptor 2 Agonists in Ovariectomized Ewes Biol Reprod, August 1, 2007; 77(2): 320 - 328. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ben Said, D. Lomet, D. Chesneau, L. Lardic, S. Canepa, D. Guillaume, C. Briant, C. Fabre-Nys, and A. Caraty Differential Estradiol Requirement for the Induction of Estrus Behavior and the Luteinizing Hormone Surge in Two Breeds of Sheep Biol Reprod, April 1, 2007; 76(4): 673 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Smith, C. M. Clay, A. Caraty, and I. J. Clarke KiSS-1 Messenger Ribonucleic Acid Expression in the Hypothalamus of the Ewe Is Regulated by Sex Steroids and Season Endocrinology, March 1, 2007; 148(3): 1150 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Taylor, M.-L. Goubillon, K. D. Broad, and J. E. Robinson Steroid Control of Gonadotropin-Releasing Hormone Secretion: Associated Changes in Pro-Opiomelanocortin and Preproenkephalin Messenger RNA Expression in the Ovine Hypothalamus Biol Reprod, March 1, 2007; 76(3): 524 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Robinson Prenatal programming of the female reproductive neuroendocrine system by androgens. Reproduction, October 1, 2006; 132(4): 539 - 547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pompolo, A. Pereira, K. M. Estrada, and I. J. Clarke Colocalization of Kisspeptin and Gonadotropin-Releasing Hormone in the Ovine Brain Endocrinology, February 1, 2006; 147(2): 804 - 810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Arreguin-Arevalo and Terry. M. Nett A Nongenomic Action of Estradiol as the Mechanism Underlying the Acute Suppression of Secretion of Luteinizing Hormone in Ovariectomized Ewes Biol Reprod, January 1, 2006; 74(1): 202 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kinoshita, H. Tsukamura, S. Adachi, H. Matsui, Y. Uenoyama, K. Iwata, S. Yamada, K. Inoue, T. Ohtaki, H. Matsumoto, et al. Involvement of Central Metastin in the Regulation of Preovulatory Luteinizing Hormone Surge and Estrous Cyclicity in Female Rats Endocrinology, October 1, 2005; 146(10): 4431 - 4436. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. McManus, R. L. Goodman, N. V. Llanza, M. Valent, A. B. Dobbins, J. M. Connors, and S. M. Hileman Inhibition of Luteinizing Hormone Secretion by Localized Administration of Estrogen, but not Dihydrotestosterone, Is Enhanced in the Ventromedial Hypothalamus During Feed Restriction in the Young Wether Biol Reprod, October 1, 2005; 73(4): 781 - 789. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Gumen and M. C Wiltbank Follicular cysts occur after a normal estradiol-induced GnRH/LH surge if the corpus hemorrhagicum is removed Reproduction, June 1, 2005; 129(6): 737 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Pillon, A. Caraty, C. Fabre-Nys, D. Lomet, M. Cateau, and G. Bruneau Regulation by Estradiol of Hypothalamic Somatostatin Gene Expression: Possible Involvement of Somatostatin in the Control of Luteinizing Hormone Secretion in the Ewe Biol Reprod, July 1, 2004; 71(1): 38 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Petersen, E. N. Ottem, and C. D. Carpenter Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol Biol Reprod, December 1, 2003; 69(6): 1771 - 1778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
