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Laboratory of Neuroendocrinology, The Babraham Institute, Babraham, Cambridge, United Kingdom CB2 4AT
Address all correspondence and requests for reprints to: Dr. J. E. Robinson, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge, United Kingdom CB2 4AT. E-mail: jane.robinson{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As one index of GnRH biosynthesis, several investigators have examined the relationship between changes in GnRH messenger RNA (mRNA) expression and the LH surge in the rat. Although several studies conclude that temporally and regionally restricted increases in cellular GnRH mRNA content occur during the steroid-induced or proestrous LH surge (12, 13, 14, 15, 16), this point is still controversial (16, 17, 18). Where changes in GnRH mRNA have been reported, the precise temporal relationship between the onset of the changes in mRNA and the LH increment remains unclear. For example, some investigators (14, 15, 16) have demonstrated that GnRH mRNA content rises in the rostral preoptic area (rPOA) at least 2 h before LH levels are seen to rise, whereas others have shown that the number of detectable GnRH mRNA-expressing cells in the more caudal POA (14) or whole rPOA (13) increases with the onset of the LH surge. Hence, it remains unclear whether a common neural mechanism may operate to alter GnRH biosynthesis and GnRH secretion at the time of the preovulatory LH surge and what causal relationship exists between the increase in mRNA and secretion.
In the present study, we sought to determine whether changes in GnRH mRNA expression occur at the time of the GnRH/LH surge in the ewe and, if so, to establish the temporal relationship between GnRH biosynthesis and secretion. This animal model has particular advantages, in that under conditions of prior progesterone withdrawal, the GnRH surge is known to be initiated by rising E concentrations alone, and the profile of GnRH secretion leading up to and throughout the surge is well established. Hence, by using in situ hybridization to assess GnRH mRNA content, we have been able to examine changes in cellular GnRH transcript levels within anatomically distinct GnRH cell populations in relation to known fluctuations in GnRH secretion throughout the time of the preovulatory surge.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Each ewe was ovariectomized and run through two artificial 14-day estrous cycles using a modification of the model of Goodman and colleagues (19). The artificial luteal phase was produced by the insertion of progesterone (1 x 9% progesterone CIDR-G intravaginal device, InterAg, Hamilton, New Zealand) and E (1 x 10 mm sc 17ß-estradiol SILASTIC brand implant, Dow Corning, Midland, MI) for 11 days. The follicular phase was initiated by the removal of progesterone, followed 16 h later by the insertion of 4 x 30-mm sc 17ß-estradiol SILASTIC implants (E).
During the first artificial follicular phase, LH concentrations were monitored in jugular blood from all ewes at hourly intervals from 8–40 h after E insertion. Data from this cycle were used to predict when a LH surge would occur in the second cycle. During the second artificial follicular phase, ewes were split into two groups and received E (surge treatment) or were subjected to sham implantation (control treatment). Ewes were killed at one of five time points relative to the time of predicted LH surge onset as follows: pre-E (n = 6), immediately before E insertion; presurge (n = 5), after E insertion but 8–10 h before LH surge onset; ascending limb (n = 8), 2–6 h after LH surge onset when LH levels are rising exponentially; midpeak (n = 7), 9–12 h after surge onset when LH levels are decreasing from the surge peak; and postsurge (n = 5), when LH levels have returned to baseline. Ewes receiving the control treatment were time matched relative to their predicted time of LH surge onset, and five animals were killed at each of the time points described above.
Hourly samples of jugular blood were taken from 6 h before E insertion until the time of death to assess LH secretion profiles. In addition, jugular blood samples were taken at half-hour intervals in both ascending and midpeak groups from 2 h before death to determine more precisely the pattern of LH release. Ewes were killed by barbiturate overdose (20 mg/kg BW, iv; Lethobarb, Duphar Vet, Southampton, UK), and the brain was rapidly removed. A block of tissue approximately 1.5 cm3 containing the preoptic area and hypothalamus was dissected from the brain within 3 min of death and rapidly frozen on dry ice. Tissue was stored at -70 C before sectioning. All animal procedures were conducted under a project license (PPL 80/1037) issued by the Home Office.
GnRH in situ hybridization
Frozen brain sections (15 µm thickness) were cut in the
coronal plane from the level of the diagonal band of Broca to the
anterior hypothalamus (AHA) on a cryostat (Bright, Huntingdon, UK).
Brain sections were thaw-mounted onto Vectabond-coated slides (3
sections/slide) so that 10 sets of every 10th section taken from the
diagonal band of Broca to the AHA were compiled for each animal.
Sections were stored at -70 C until used.
A 39-mer oligonucleotide probe complementary to the GnRH-encoding region of the partially cloned ovine GnRH complementary DNA sequence (20) was used for GnRH mRNA in situ hybridization (probe sequence, 5'-TCT CTT TCC TCC AGG GCG CAG CCC ATA GGA CCA GTG CTG-3'). The six 3'-residues of the probe are complementary to those encoding the first two amino acids of GnRH and, although not yet identified in the sheep, are identical in human, mouse, and rat GnRH sequences. EMBL database searches showed no significant homology other than that of other mammalian GnRH coding sequences. The oligonucleotide was 3'-end labeled with [35S]deoxy-ATP (1000–1500 Ci/mmol; New England Nuclear-DuPont, Boston, MA) using terminal deoxynucleotidyl transferase (50 U; Pharmacia, Uppsala, Sweden) and purified by filtration on a Sephadex G-50 column, resulting in a specific activity of approximately 109 cpm/mg probe.
Because of the large number of slides involved (three or four slides per animal with five or six animals per group and five different time points), the in situ hybridization was undertaken as two separate experiments, 24 h apart. Slides from the five control group ewes underwent hybridization together as one set, whereas slides from the five E-treated groups of ewes were processed in an identical fashion the following day using the same labeled probe. Sections were quickly warmed to room temperature using a hair dryer, fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min, and rinsed in 0.1 M PBS. Sections were dehydrated through increasing ethanol concentrations of 70%, 80%, 90%, 95%, and 100% and allowed to air-dry. For hybridization, the 35S-labeled probe was diluted in hybridization buffer [20 x saline sodium citrate (SSC), 50% deionized formamide, 10% dextran sulfate, 1 x Denhardt’s solution, 250 µg/ml sheared salmon testicular DNA, and 0.3% ß-mercaptoethanol] to give a final concentration of approximately 1.2 x 103 cpm/µl, and 250 µl were applied to each slide. After overnight hybridization at 37 C, sections were washed in 1 x SSC at room temperature, three times in 1 x SSC at 55 C (30 min each), and finally in 1 x SSC for 1 h at room temperature. After a brief rinse in water, sections were placed in a 300-mM ammonium acetate-70% ethanol solution for 30 sec, followed by absolute ethanol for 30 sec, then allowed to air-dry. The slides were assorted randomly, dipped in Ilford K-5 nuclear track emulsion, and exposed for 2–3 days in light-tight boxes. All slides were developed with Ilford Phenisol (diluted 1:5 in distilled water; 5 min at 20 C) and counterstained lightly with methylene blue. Competition experiments were undertaken in which the 35S-labeled probe was applied to the brain sections in the presence of a 50-fold excess of unlabeled probe.
Analysis
The number of cells expressing GnRH mRNA per section was
determined for each animal by counting the total number of positively
hybridized cells located within anatomically matched sections
containing the rPOA and medial septum (MS). Cells were considered to be
positively hybridized when silver grains were found clustered over a
methylene blue-counterstained cell body, and the number of silver
grains was greater than 5 times the number counted over preoptic cells
in the excess cold oligo controls (21, 22). Hybridized cells were
assigned to either the rPOA or MS depending on their anatomical
location, as revealed by the methylene blue counterstaining.
Specifically, all hybridized cells clustered around the organum
vasculosum of the lamina terminalis (OVLT) and the rostral extension of
the supraoptic recess of the third ventricle were classified as rPOA
GnRH neurons (Fig. 3
, B–D). Cells found rostral to the OVLT (Fig. 3A)
were few in number and, therefore, were excluded from the analysis.
Hybridized cells lying dorsal to this population, in the midline, and
surrounded by the morphologically distinct magnocellular neurons of the
MS, were termed MS GnRH neurons (Fig. 3, C and D). As insufficient
material existed with which to accurately match all groups, the
hybridized cells found more caudally in the ventrolateral aspect of the
anterior hypothalamus were not analyzed. Cell counts from three or four
sections for each GnRH population were used to provide individual
animal means and then combined to provide treatment group means and
SE values at each time point.
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Statistical analysis of cell numbers and silver grain density between time points within the control and E-treated experimental groups were assessed using ANOVA with Tukey’s post-hoc test. Significance was set at P < 0.05.
LH RIA
Plasma LH was measured in duplicate 100-µl aliquots using a
previously described, double antibody RIA procedure with the anti-LH
antiserum CSU 204 (G. D. Niswender, Fort Collins, CO) and the
NIDDK S11 standard (23). Inter- and intraassay coefficients of
variation were 13.24% and 8.67%, respectively, for six assays. The
average detection limit of the assay was 0.195 ng/ml. The time of onset
of the LH surge was defined as the first sample of a sustained rise in
LH levels that was greater than the mean ± 2 SD of the five
preceding samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, right panel) and surge treatments (Fig 2, left panel). Due to
within-animal variation, not all animals in the ascending limb and
midpeak groups were killed at the appropriate times. Ewes in these
groups were reallocated, based on individual LH data, resulting in both
the ascending limb and midpeak groups containing five animals each. The
five remaining animals from these two groups were killed before the LH
surge began and thus were excluded from analysis.
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and 3, C and D). Smaller numbers of hybridized cells
were also found in the midline above and rostral to the rPOA in the MS
(Figs. 2B and 3, C and D).
Effects of E treatment on hybridized cell number
No significant differences (P > 0.05, by ANOVA)
were evident in the number of hybridized cells detected in the rPOA or
MS of control or E-treated group across the five time points (Table 1).
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). In
contrast, a significant change in mean cellular silver grain density
over the five time points was found in both the rPOA and MS of
E-treated ewes (P < 0.05, by ANOVA). Specifically, a
significant decrease in cellular silver grain density occurred between
the presurge and ascending limb time points in both the rPOA and MS
(rPOA cells, 0.64 ± 0.06 vs. 0.43 ± 0.05 silver
grain density in arbitrary units/µm2; MS cells, 1.08
± 0.09 vs. 0.77 ± 0.07; P < 0.05, by
Tukey’s post-hoc test). This was preceded by a
nonsignificant increase between pre-E and presurge time points. Silver
grain density remained relatively constant over the ascending limb,
midpeak, and postsurge time points in both rPOA and MS cells (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study has examined the temporal changes in cellular GnRH mRNA expression that are associated with the E-induced LH surge in the ewe. Our results show that significant changes in GnRH mRNA expression occur in ewes before the onset of the estradiol-induced LH surge, whereas time-matched control animals exhibit no changes. Specifically, cellular GnRH mRNA content was found to fall between the presurge and ascending limb time points of our study, which represents an approximately 8-h period of time over which circulating LH concentrations change from basal levels to the initial increment at the start of the LH surge. Recent work has shown that the latter portion of this period represents a time when a gradual increase in GnRH pulse frequency and amplitude coupled with elevated GnRH interpulse secretion occur before the onset of the GnRH surge (11). Hence, our findings indicate that in the ewe, GnRH mRNA levels fall over a period when GnRH secretion is increasing gradually before the onset of the GnRH surge.
It is also of interest to note that GnRH mRNA remained constant over the ascending limb, midpeak, and postsurge time points. Using an animal model similar to that used in this study, Moenter and colleagues (26) reported that Fos expression (a marker of neuronal activation) was increased in GnRH neurons during the GnRH surge. As the maximum number of Fos-positive neurons occurs after the initiation of the surge, these authors suggest that Fos induction may be related to the replenishment of GnRH in neurons requiring an increase in cellular GnRH mRNA. Our data do not support this view. Thus, it does not appear that the surge-related increase in Fos leads to an acute increase in cellular GnRH mRNA content.
Species comparisons
These observations in the sheep appear to be in contrast to
those reported in the rat. Thus, where changes in GnRH mRNA expression
are reported these constitute an increase in mRNA that is closely
associated with the LH surge (12, 13, 14, 15, 16). In terms of the absence of an
increase in cellular GnRH mRNA content in the ewe, we did note a
nonsignificant increase in GnRH mRNA expression after estrogen
administration in the presurge group before the significant fall in
mRNA content. Hence, it is possible that a rise in GnRH mRNA expression
also occurs in the ewe, but does so many hours before the preovulatory
changes in GnRH secretion. Perhaps a larger sample size would have
allowed us to make this determination. However, because of the
necessity in this study to examine GnRH mRNA content in the ewe at time
points separated by relatively long time intervals, it is possible that
we may have missed a peak of GnRH mRNA expression. Future studies will
focus on the time period following the administration of E, but before
the GnRH surge begins.
In terms of the temporal relationship between the change in GnRH mRNA and the GnRH surge, there would appear to be a clear species difference. The reason for this is unclear. It is, however, worth noting that the sheep model used in these studies is more physiological than the E-induced LH surges produced in some of the rat studies. In the latter case the LH surge is reduced in amplitude and delayed compared with a surge produced by estrogen and progesterone. Other factors that may be relevant are the differences between rats and sheep in the length of the ovarian cycle, the lack of a true luteal phase in the rat, and the degree to which a circadian input is used to trigger the onset of the surge (27, 28). Hence, although we can only speculate about why the profile of GnRH mRNA changes leading up to the LH surge are different between rats and sheep, one possibility is that the circadian trigger plays a role in generating the mRNA increase as well as synchronizing it with the neural apparatus responsible for GnRH secretion at the time of the surge.
Relationship between GnRH secretion and biosynthesis
As the changes in mRNA biosynthesis do not occur concomitantly
with the onset of GnRH surge secretion in the ewe, our results suggest
that different mechanisms may underlie these phenomena. Although the
GnRH surge clearly represents the stimulatory actions of E on the GnRH
neurosecretory system, the reduction in GnRH mRNA that we have observed
may be related more to the negative feedback effects of E on GnRH
secretion that precede the GnRH surge in this species (10, 11). At the
least, it would seem that the effects of estrogen on GnRH mRNA
expression and secretion are uncoupled in the hours leading up to the
surge as mRNA content falls while pulsatile and interpulse GnRH
secretion increases. Although, the direction of change in GnRH mRNA and
secretion is the same in the rat, the temporal differences between the
onset of the two events reported by some investigators have similarly
led them to suggest that the neural mechanisms underlying the secretory
and mRNA changes in the rat may also be different (15).
As GnRH neurons in the ewe (29, 30) and other species (31, 32, 33) do not contain classical nuclear estrogen receptors, estrogen-receptive interneuron populations may play a role in transmitting the estrogen signal to GnRH neurons. Recent work has shown that estrogen receptor-containing neurons located in the arcuate nucleus project to the median eminence (34), whereas estrogen-receptive cells located in the POA as well as the mediobasal hypothalamus project to the vicinity of the GnRH cell bodies in the rPOA of the ewe (Herbison, A. E., and A. Caraty, unpublished observation). These observations suggest that estradiol may have the capacity to modulate GnRH biosynthesis and secretion independently through different neuronal populations that target the GnRH cell body or terminal, respectively. If so, these different neural pathways, which have only been partially defined in terms of their neurotransmitter content (35), may underlie the differential regulation of GnRH neurons by estrogen.
The mechanisms underlying the changes in GnRH mRNA identified in this study are not known. Although the influence of estrogen on GnRH mRNA content in the rat is known to involve increases in GnRH gene transcription (16, 36), there is increasing evidence that supports the posttranscriptional regulation of GnRH mRNA content in vivo (37). In the present study we have identified a decrease in GnRH mRNA content. As changes in the stability of GnRH mRNA can be relatively rapid (38, 39), it is possible that this might result from increased degradation of the message. Such a conclusion awaits further studies.
Conclusion
In summary, this study has evaluated the changes in GnRH mRNA
expression associated with generation of the GnRH surge in the ewe.
That a significant decrease in expression is reported before the GnRH
surge suggests that changes in GnRH biosynthesis precede the massive
and sustained release of GnRH in this species and that GnRH mRNA
expression may be declining at a time when GnRH secretion increases.
The significance of these changes in GnRH mRNA expression to the
successful generation of the GnRH surge warrants further
investigation.
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Acknowledgments |
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Footnotes |
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2 Present address: Reproductive Sciences Program, University of
Michigan, Ann Arbor, Michigan 48109. Medical Research Council-funded
graduate student.
3 Present address: I.N.R.A., Physiologie de la Reproduction des
Mammifères Domestiques, 37380 Nouzilly, France. Research Fellow
of St. Catharine’s College (Cambridge, UK).
4 Lister Institute-Jenner Fellow.
Received July 3, 1997.
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