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Department of Psychology and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824
Address all correspondence and requests for reprints to: Dr. Cheryl L. Sisk, Department of Psychology, Michigan State University, East Lansing, Michigan 48824. E-mail: sisk{at}pilot.msu.edu
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Abstract |
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Introduction |
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In several species, the signal to initiate LH and, presumably, LHRH release at puberty onset involves a decrease in responsiveness to negative feedback by gonadal steroids. In prepubertal ferrets (14, 15), sheep (1), and hamsters (16), gonadal steroids are particularly strong inhibitors of gonadotropin secretion, but at puberty onset, the negative feedback action of steroids is greatly diminished, resulting in an increase in pulsatile LH release. Although the mechanisms underlying the change in the set-point of this neuroendocrine feedback loop are unknown, they presumably involve a change in the response of steroid hormone target cells that either directly or indirectly restrain LHRH release in prepubertal animals.
This experiment had two objectives: 1) to determine whether populations of LHRH neurons in specific brain regions contribute to the pubertal increase in LHRH secretion, and 2) to test the hypothesis that the pubertal decrease in responsiveness to steroid negative feedback regulation of LHRH is mediated by differential effects of testosterone on subpopulations of LHRH neurons in prepubertal and postpubertal males. In situ hybridization was used to detect LHRH mRNA within neurons in male ferrets at prepubertal and postpubertal ages in both the presence and the absence of gonadal steroid hormones. We reasoned that any age-related increases in LHRH mRNA levels within LHRH neurons would reflect participation in the pubertal increase in LHRH release, as those neurons would need to replenish their releasable pools of LHRH. If such increases in LHRH mRNA were restricted to or most prevalent in subpopulations of LHRH neurons within specific brain regions, then these cells would be implicated in the pubertal increase in pulsatile release of LHRH. Similarly, if manipulation of gonadal steroids exerted influences on steady state levels of LHRH mRNA in a brain region-specific manner, inferences could be made about subpopulations of LHRH neurons that are regulated by steroid feedback in prepubertal and postpubertal males. The ferret is a particularly interesting animal model for the study of brain region specificity in the regulation of LHRH because the distribution of LHRH neurons in this species includes not only cell bodies in the rostral forebrain, but also a substantial population of LHRH neurons within cell groups in the mediobasal hypothalamus (17, 18, 19). Therefore, this experiment includes an analysis of LHRH mRNA in cells in the preoptic area (POA), retrochiasmatic area (RCH), arcuate nucleus (ARC), and median eminence (ME).
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Materials and Methods |
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Blood and brain tissue collection
At either 12 or 24 wk of age, ferrets from the three treatment
groups (intact, castrate+oil, castrate plus TP) were deeply
anesthetized with Equithesin anesthetic (2.5 ml/kg, ip), and a terminal
blood sample was collected via cardiac puncture into a heparinized
tube. Blood samples were centrifuged, and plasma was removed and stored
at -20 C until used in a RIA for testosterone. Animals were perfused
intracardially with 350 ml heparinized 0.1 M PBS, followed
by 350 ml 4% paraformaldehyde in 0.1 M PBS. Brains were
removed and stored at -70 C until sectioning. Coronal brain sections
(20 µm) were cut on a cryostat, thaw-mounted onto
poly-L-lysine-coated slides, and vacuum-desiccated
overnight. Slides were stored in boxes containing desiccant at -70 C
until in situ hybridization was performed.
RIA of testosterone
Plasma concentrations of testosterone were measured in the
terminal plasma samples with a Coat-a-Count Total Testosterone Kit
(Diagnostic Products Corp., Los Angeles, CA). The minimum detectable
concentration for this assay was 0.11 ng/ml, and the coefficient of
variation was 13.0%.
In situ hybridization
Every 12th brain section from each animal was used for in
situ hybridization for LHRH mRNA. All tissue was run together in a
single assay. In situ hybridization was performed using a
48-base synthetic oligonucleotide complementary to the LHRH-coding
region (bases 102–149) of the human complementary DNA (21). This
oligomer was synthesized at the Michigan State University
Macromolecular Structure Facility on an Applied Biosystems 380B DNA
synthesizer (Foster City, CA) and purified with a C18
Sep-Pak cartridge (Waters Associates, Milford, MA). The probe (15
pmol/µl) was 3'-end labeled by incubation for 60 min at 37 C with
[35S]deoxy-ATP (75 pmol; 1,300 Ci/mmol; New England
Nuclear, Boston, MA) and terminal deoxynucleotidyl transferase (25 U;
Boehringer Mannheim, Indianapolis, IN) to a specific activity of about
106 cpm/µl. The hybridization protocol was modified
slightly from published methods for detection of LHRH mRNA (22, 23, 24).
Prehybridization treatment of tissue consisted of warming the sections
to room temperature, incubation for 30 min in 0.001% proteinase K at
37 C, followed by incubation in 0.0025% acetic anhydride in 0.1
M triethanolamine (pH 8.0). Sections were rinsed briefly in
2 x NaCl-Na citrate (SSC), dehydrated through a series of
ethanols, and dried in a vacuum desiccator. A saturating concentration
of LHRH complementary DNA probe was determined by applying varying
amounts of 35S-labeled probe, ranging from
50,000–1,000,000 cpm/slide, to test sections. Based on the saturation
curve generated from this assay (Fig. 1
),
500,000 cpm labeled probe in 100 µg/ml yeast transfer RNA, 500
µg/ml salmon sperm DNA, and Denhardt’s solution (0.02% Ficoll,
0.02% polyvinylpyrrolidone, and 0.02% BSA) were applied to each
slide. Slides were coverslipped with parafilm, and hybridization
proceeded for 24 h at 37 C. Coverslips were then removed, and
sections were rinsed in 4 x SSC and desalted in decreasing
concentrations (1 and 0.5 x) of SSC containing 1.0 M
dithiothreitol (DTT; 0.1%), followed by 30-min washes in 0.1 x
SSC containing 1.0 M DTT at 42 C. The posthybridization
wash temperature was based on analysis of a separate melting curve
assay, in which posthybridization temperatures ranged from 37–75 C. In
the melting curve analysis, no labeled cells were found in brain
sections that were incubated at temperatures higher than 65 C. A final
wash in 0.1 x SSC containing 1.0 M DTT was performed
at room temperature. The slides were dehydrated through a series of
ethanols and dipped in emulsion (NTB-3, Eastman Kodak, Rochester, NY)
diluted 1:1 with distilled water. After a 3-week exposure period,
slides were developed in Kodak D-19 developer and counterstained with
methylene blue. Preincubation of the tissue with an unlabeled probe or
preincubation with 20 µg/ml ribonuclease A completely abolished
labeling.
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. The majority of LHRH
neurons in the ferret brain, as identified by immunocytochemistry, is
found within these four brain regions, with the remainder diffusely
scattered throughout other brain areas, primarily the lateral
hypothalamus, diagonal band of Broca, and septum (19). All sections
containing POA, RCH, ARC, and ME were analyzed, and the mean
(±SEM) numbers of sections per area for each brain were
7.0 ± 0.1, 6.1 ± 0.1, 9.9 ± 0.2, and 9.9 ± 0.2,
respectively. The number of silver grains overlying cells in these
brain regions was quantified with a Bioquant Image Analysis System Meg
IV (R&M Biometrics, Nashville, TN). Image analysis was performed using
brightfield optics at x1000 magnification by an experimenter blind to
age and treatment group. A threshold value was set so that the silver
grains overlying the cell were above and the underlying cell body was
below the threshold. The cell body was circumscribed, and the area of
the cell body and the number of pixels representing silver grains were
determined. The trace circumscribing the cell was then moved to an
adjacent area of neuropil, and the number of pixels representing silver
grains within this area of background was obtained. Cells with 5 or
more times the number of silver grain pixels in the adjacent background
area were classified as labeled.
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5 times background grains criterion)
was analyzed by three-way ANOVA (pubertal status by treatment by brain
region). Second, the intensity of labeling was analyzed by performing
three-way ANOVA on the mean number of silver grain pixels per labeled
cell for each individual (pubertal status by treatment by brain
region). Scheffe’s F test was used for post-hoc
comparisons. In all analyses, P 
0.05 was considered
significant. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Post-hoc comparisons
confirmed the expected pubertal increase in testosterone in intact
males, but also revealed that testosterone treatment unexpectedly
resulted in significantly higher testosterone concentrations in
postpubertal castrates than in prepubertal castrates.
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shows examples of cells in the ARC that
were labeled by in situ hybridization using a 5 or more
times background silver grains criterion. There was a significant
three-way interaction among brain region, treatment, and pubertal
status on the number of labeled cells (F(6,85) = 3.56;
P = 0.003), indicating that any two independent
variables produced a different pattern of results at different levels
of the third variable. Therefore, the data were further analyzed by
examining the effects of pubertal status and treatment separately for
each brain region (Fig. 5).
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). Two
important features of this interaction were revealed by
post-hoc analysis. First, the number of labeled cells in the
ARC increased with pubertal maturation, but only in intact males.
Second, castration resulted in a significant increase in the number of
labeled cells in the ARC of prepubertal, but not postpubertal,
males.
In the RCH, there was also a significant interaction between pubertal
status and treatment, but the pattern of results was different from
that in the ARC (F(2,22) = 5.53; P = 0.011;
Fig. 5). First, there was no pubertal change in the number of labeled
cells within any treatment group. Second, manipulation of gonadal
steroids affected the number of labeled cells only in postpubertal
males, in which testosterone treatment resulted in a greater number of
labeled cells than that in intact males.
Although the overall pattern of results in the ME was similar to that
in the ARC, there were no significant effects of either pubertal status
or treatment on the number of labeled cells (Fig. 5). In the POA, the
number of labeled cells was not affected by either pubertal status or
treatment, nor was there an interaction between pubertal status and
treatment (Fig. 5).
Labeling intensity within labeled cells
The mean number of pixels representing silver grains over labeled
cells is shown for all treatment groups and brain regions in Fig. 6. Statistical analysis showed no
three-way interaction among pubertal status, treatment, and brain
region on this measure. There was a significant main effect of
treatment (F(2,79) = 4.996; P = 0.01) that
did not interact with either pubertal status or brain region. Because
of the lack of interactions between treatment and the other two
variables, post-hoc analysis was performed after collapsing
data across brain region and pubertal status. In this analysis,
labeling intensity per cell was significantly less in
testosterone-treated castrates than that in oil-treated castrates.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Brain region-specific regulation of LHRH mRNA during puberty
It is clear from the present experiment that of the four
populations of LHRH neurons studied, LHRH mRNA increases most
dramatically in the ARC as intact males go through puberty. There was
no change in either the number of labeled cells or in labeling
intensity in ME, POA, or RCH after pubertal maturation. We cannot rule
out the possibility that there were changes in LHRH mRNA levels in
these brain regions within cells that had less than 5 times the number
of background silver grains. At the very least, however, pubertal
alterations in LHRH mRNA are not as pronounced in the ME, POA, and RCH,
implying that LHRH neurons in the ARC are the principal population of
LHRH cells involved in pubertal maturation of the reproductive
neuroendocrine axis.
The pubertal increase in the number of labeled cells in the ARC could either reflect an increase in the amount of mRNA per cell, so that cells expressing low levels before puberty meet the labeling criterion after puberty, or could indicate an increase in the number of cells that are phenotypically LHRH. Although it is difficult to resolve this question, we favor the former interpretation, because when LHRH neurons in the rat are visualized by immunocytochemical procedures optimized to detect the maximal number of LHRH cells, the number of LHRH neurons is constant from early postnatal ages through young adulthood (8).
The results of this experiment help to resolve questions about the interpretation of previous data on the role of arcuate LHRH neurons during puberty in the ferret. In an immunocytochemical study, we found that the number of LHRH-immunopositive cells in the ARC was approximately 50% lower in postpubertal males than in prepubertal males (19). Similar to the present experiment, the pubertal change in LHRH immunoreactivity was most pronounced in the ARC. Colchicine treatment of two adults in this study did not result in increased numbers of ARC LHRH neurons, suggesting that the pubertal reduction in immunoreactivity was not secondary to depletion of cell body stores in actively secreting neurons (19), but instead that some ARC neurons cease production of LHRH in adult animals. However, in light of the present demonstration that ARC LHRH mRNA levels dramatically increase with puberty (the data from the two colchicine-treated ferrets notwithstanding), it now appears more likely that the pubertal decrease in the number of immunoreactive LHRH cells in the ARC is a reflection of decreased and undetectable LHRH cell body stores in actively secreting arcuate neurons of the postpubertal male. Although we cannot exclude either the possibility that the pubertal increase in LHRH mRNA within ARC neurons is not accompanied by an increase in LHRH protein or that the pubertal decrease in immunoreactivity does reflect decreased protein synthesis, the increase in LH pulse frequency that is characteristic of puberty strongly implies that both LHRH synthesis and release would need to be increased.
A large reduction in the number of immunopositive LHRH cells occurs after mating in the female ferret and is thought to reflect a depletion of cell body stores of LHRH as a result of the mating-induced LHRH surge (25). In this case, the reduction in the number of immunoreactive cells occurs in rostral brain regions as well as in the ARC. In addition, fos is expressed within LHRH neurons in both the POA and ARC in association with the mating-induced LH surge in the female ferret (26, 27). These studies together with those of the present study suggest that LHRH neurons in a wider range of brain regions are involved in the production of a mating-induced LH surge in females compared with those involved in the pubertal increase in LH pulse frequency in males. Evidence that the spontaneous preovulatory LHRH surge in rodents is also characterized by a relatively widespread activation of LHRH neurons has been previously reported (28).
The increase in LHRH mRNA levels in postpubertal males observed in this experiment generally agrees with reports of increases in LHRH mRNA during postnatal development in the rat, in which LHRH mRNA levels appear to rise in advance of increases in LH release (5–7; but see Ref.29). The present study was not designed to determine when pubertal increases in LHRH mRNA occur relative to the pubertal increase in LH pulse frequency that occurs in male ferrets (15). Thus, the observed increase in LHRH mRNA in the ARC of postpubertal male ferrets could either precede pubertal activation of neurosecretion or be a consequence of pubertal activation of arcuate LHRH cells. That is, LHRH synthesis could increase to replenish released stores in these cells. We also do not know whether the changes in steady state levels of LHRH mRNA in this study reflect an increase in the rate of transcription or an increase in the stability of the mRNA. However, the increase in steady state levels of LHRH mRNA that occurs just before puberty in the female rat is probably due to an increase in mRNA stability, as levels of LHRH primary transcript increase only at a much earlier age (7).
Brain region-specific regulation of LHRH mRNA by testosterone
Two important conclusions about the regulation of LHRH mRNA by
testosterone can be drawn from this experiment. First, manipulation of
testosterone exerts brain region-specific effects on LHRH mRNA
levels, and second, the brain region most affected by manipulation of
testosterone varies with reproductive maturity. Castration of
prepubertal males increased the number of labeled cells only in the
ARC, indicating that of the entire population of LHRH neurons, those in
ARC are most strongly regulated by gonadal steroids in prepubertal
males. In contrast, the most dramatic effect of castration in
postpubertal males was observed in the POA, in which there was a
pronounced increase in labeling intensity, indicating that these cells
are involved in steroid negative feedback regulation of LHRH in
adulthood. Thus, there appears to be a developmental shift in the
populations of LHRH neurons that are most strongly regulated by gonadal
steroid negative feedback. Such a shift could be part of the mechanism
underlying the pubertal decrease in responsiveness to steroid negative
feedback, because LHRH neurons in the POA comprise a much smaller
population than those in the ARC, and the two populations of cells
exist within different local environments (e.g. afferent
input and glial interactions).
In the RCH, testosterone treatment of castrated adults resulted in a greater number of labeled cells compared with that in intact adults; a similar effect was not observed in prepubertal males. The higher circulating concentrations of testosterone produced by the daily injections in adults may have exerted pharmacological actions that indirectly stimulated LHRH mRNA levels in this subpopulation of LHRH cells. In all other brain regions examined, the numbers of labeled cells in testosterone-treated prepubertal and postpubertal castrates were similar, indicating that the difference in circulating levels of testosterone produced by the injections did not exert differential effects on this measure. However, given that prepubertal males are more responsive to the negative feedback effects of testosterone (15), it is possible that additional differences between testosterone-treated prepubertal and postpubertal males would have been observed had circulating concentrations of testosterone been the same in these two groups.
Previous studies of the regulation of LHRH mRNA by testosterone in male rats have not provided a clear picture. Levels of LHRH mRNA have been reported to increase (22, 30, 31), decrease (32), or not change (33, 34, 35) after castration. Numerous methodological differences could account for these discrepancies, including postcastration time interval, method of detecting LHRH mRNA, and species used. Nevertheless, it appears that at least under certain conditions, LHRH gene expression is affected by gonadal hormones. The present study indicates that effects of testosterone on LHRH mRNA are exerted differentially on specific populations of LHRH neurons, and, importantly, that these subpopulations change during reproductive maturation.
Proposed organization of the LHRH neuronal system
Together with our earlier immunocytochemical data, the present
experiment suggests a framework for the organization of the LHRH
neuronal system in male ferrets. We propose that when LHRH release is
increased in young males, either as they go through puberty or in
response to castration, LHRH neurons in the ARC are called upon to
respond to the demand for increased LHRH release. This proposal is
based on the facts that the pubertal reduction in LHRH immunoreactive
cells, which we believe to reflect increased release, is most
pronounced in the ARC (19), and that levels of LHRH mRNA are increased
most prominently in the ARC after puberty or after castration of
prepubertal males (present experiment). In adults, LHRH neurons in the
ARC may already be functioning at or near capacity, so that when adults
are castrated, the increased demand for LHRH release is met by
increasing LHRH mRNA within LHRH neurons of the POA. However, a similar
effect of castration on LHRH mRNA across brain regions would be
predicted in prepubertal and adult males if the degree to which LHRH
mRNA is increased is simply proportional to the demand for LHRH
release, because LH pulse frequency is similar in prepubertal and adult
castrates (15). Thus, an important additional feature of this
organizational framework is that, as a consequence of pubertal
maturation, there is a shift in the particular population of LHRH
neurons in which LHRH mRNA is most strongly regulated by gonadal
steroids. Such a shift could underlie pubertal changes in
responsiveness to negative feedback effects of testosterone on the
hypothalamic-pituitary axis.
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Acknowledgments |
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Footnotes |
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2 Present address: Institute of Biomedical Sciences, Academia Sinica,
Taipei, Taiwan, Republic of China.
3 Present address: Department of Physiology, University of Kentucky,
Lexington, Kentucky 40536.
Received May 28, 1997.
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