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Endocrinology Vol. 138, No. 11 4740-4747
Copyright © 1997 by The Endocrine Society

ARTICLES

Brain Region-Specific Regulation of Luteinizing Hormone-Releasing Hormone Messenger Ribonucleic Acid in the Male Ferret: Interactions between Pubertal Maturation and Testosterone1

Yu Ping Tang2, Michael L. Kashon3 and Cheryl L. Sisk

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study examined the regulation of LHRH messenger RNA (mRNA) during pubertal maturation and by testosterone in male ferrets. Prepubertal and postpubertal ferrets were either intact or were castrated and treated with daily injections of oil or 5 mg/kg testosterone propionate for 14 days. In situ hybridization for LHRH mRNA was performed using an 35S-labeled 48-base oligonucleotide complementary to the human LHRH-coding region. Computerized image analysis was performed on cells in the preoptic area, retrochiasmatic area, arcuate nucleus (ARC), and median eminence; cells were classified as labeled if the number of pixels representing silver grains over the cell was 5 or more times the number of background silver grain pixels. Both pubertal maturation of intact males and castration of prepubertal males resulted in an increase in the number of labeled cells in the ARC. These effects were not observed in any of the other three brain regions, suggesting that ARC LHRH-producing neurons are of primary importance in the presumed increase in LHRH release that occurs as a consequence of either pubertal maturation or castration of prepubertal males. Castration of adults did not increase the number of labeled cells in any brain area, but resulted in an increase in silver grains per labeled cell only in the preoptic area. Thus, LHRH mRNA is regulated during puberty primarily in the ARC, and the particular cell group in which LHRH mRNA is most strongly regulated by testosterone changes with pubertal maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ONSET of puberty requires activation of the neurons that synthesize and release LHRH. An increase in LHRH secretion during puberty stimulates gonadotropin secretion and subsequent gonadal maturation (reviewed in Refs. 1–4). LHRH neurons synthesize their peptide hormone well before the onset of puberty, as evidenced by the presence of LHRH messenger RNA (mRNA) and protein within the brain at early postnatal ages (5, 6, 7, 8, 9). In addition, administration of glutamate receptor agonists to prepubertal animals can elicit LHRH release and induce early puberty (10, 11, 12, 13). Thus, although LHRH neurons in prepubertal animals are capable of releasing LHRH in a manner sufficient to sustain fertility, they are apparently awaiting the appropriate signals to do so.

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).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
All protocols involving animals were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Michigan State University committee on animal use and care. Weanling male European ferrets (Mustela putorius furo) were purchased from Marshall farms (North Rose, NY) at 7 weeks of age. They were housed in the laboratory in stainless steel cages (51 x 60 x 38 cm) with Purina ferret chow (Ralston-Purina, St. Louis, MO) and water available ad libitum. Ambient temperature in the colony room was maintained at 23 ± 1 C, and the initial light-dark cycle was 8 h of light and 16 h of darkness (LD 8:16). A 2 x 3 experimental design was used that resulted in three treatment groups (intact, castrate, and testosterone-treated castrate animals) at 2 different stages of reproductive maturity (prepubertal and postpubertal, n = 14 of each age). Of the 14 prepubertal subjects, 9 ferrets were castrated at 8 weeks of age while under methoxyflurane anesthesia (Metofane, Pitman-Moore, Washington Crossing, NJ), and 5 ferrets remained intact. Beginning 2 weeks after castration, 5 castrates received daily injections of testosterone propionate (TP; 5 mg/kg, sc) for 2 weeks, and the remaining 4 castrates received daily injections of oil vehicle. At the end of the 2-week treatment period, when the ferrets were 12 weeks old, these treated ferrets and the 5 prepubertal intact animals were perfused, and brain tissue was collected as described below. When the 14 ferrets assigned to postpubertal treatment were 12 wk old, the light-dark cycle was changed from LD 8:16 (short days) to LD 18:6 (long days). This photoperiod manipulation was used to induce and synchronize the onset of puberty in this group of males, because the age at puberty onset in males raised to adulthood in short days is quite variable. However, once adulthood has been reached, parameters of LH secretion are similar in males that have undergone puberty in either short days or after a transition from short to long days (20). At 20 weeks of age, when full testicular size had been reached, 9 of these postpubertal ferrets were castrated, and 5 remained intact. Beginning 2 wk after castration, 5 castrates received daily injections of TP (5 mg/kg, sc), and 4 received daily injections of oil vehicle for 2 weeks. At the end of the 2-week treatment period (24 wk of age), these ferrets and the remaining 5 intact postpubertal males were perfused as described below.

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. 1Go), 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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  		Figure 1. Mean (± SEM) number of pixels representing silver grains overlying cells labeled after in situ hybridization for LHRH mRNA with increasing concentrations of oligoprobe. Based on this saturation analysis, 500,000 cpm labeled probe were applied to each slide in this experiment.

 
Microscopic analysis
Quantification of silver grains was performed using microscopic image analysis of cells in four brain regions: the POA, RCH, ARC, and ME. These brain regions were operationally defined as shown in Fig. 2Go. 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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  		Figure 2. Line drawings of coronal sections through the ferret POA and hypothalamus. Hatched areas show operational definitions of the POA, RCH, ARC, and ME; image analysis was performed on cells within these areas to determine the number of cells labeled after in situ hybridization for LHRH mRNA.

 
Data analysis
Plasma concentrations of testosterone were analyzed by two-way ANOVA (pubertal status by treatment). Quantitative analysis of in situ hybridization was analyzed in two ways. First, the number of labeled cells (as defined by the >=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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma concentrations of testosterone
There was a significant interaction between pubertal status and treatment on plasma concentrations of testosterone (F(2,21) = 5.187; P < 0.05; Fig. 3Go). 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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  		Figure 3. Mean (± SEM) plasma testosterone concentrations in intact, castrated, or testosterone-treated castrated prepubertal and postpubertal ferrets. Brackets designate significant differences between treatment groups within an age, and asterisks designate significant differences between age groups within a treatment group.

 
Brain regional analysis of number of labeled cells
The overall distribution of cells labeled after in situ hybridization for LHRH mRNA was the same as that for cells labeled after immunocytochemistry for LHRH protein (19). In the rostral forebrain, labeled cells were sparsely distributed through the diagonal band of Broca, septum, and POA. More caudally, a somewhat larger population of cells was found at the base of the hypothalamus in the RCH, and the largest population of cells was located in the ventral aspect of the ARC, with some cells within the ME proper. Figure 4Go 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. 5Go).



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  		Figure 4. Photomicrograph of the ARC of an intact male ferret after in situ hybridization for LHRH mRNA. Arrows indicate labeled cells (>=5 times the background silver grains). 3V, Third ventricle. Bar = 100 µm.

 


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  		Figure 5. Mean (± SEM) number of cells labeled after in situ hybridization for LHRH mRNA in the POA, RCH, ARC, and ME of intact, castrated, and testosterone-treated castrated prepubertal and postpubertal male ferrets. Brackets designate significant differences between treatment groups within an age, and asterisks designate a significant difference between age groups within a treatment group.

 
Within the ARC, manipulation of gonadal steroids produced a different pattern of results in prepubertal and postpubertal males (i.e. a pubertal status by treatment interaction; F(2,22) = 4.4; P = 0.025; Fig. 5Go). 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. 5Go). 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. 5Go). 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. 5Go).

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. 6Go. 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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  		Figure 6. Mean (± SEM) number of pixels representing silver grains over cells labeled after in situ hybridization for LHRH mRNA in the POA, RCH, ARC, and ME of intact, castrated, and testosterone-treated castrated prepubertal and postpubertal male ferrets.

 
There was a significant interaction between pubertal status and brain region (F(6,79) = 3.98; P = 0.011) on labeling intensity, and post-hoc comparisons were made after collapsing data across treatment groups. In this case, labeling intensity was greater in postpubertal males than in prepubertal males, but only for cells in the POA and ARC. Within the POA, this effect appeared to be due primarily to the rather high intensity of labeling in adult castrates, whereas in the ARC, labeling intensity was somewhat higher in all three adult groups compared with that in the three prepubertal groups (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This experiment demonstrates that levels of LHRH mRNA in the male ferret brain are affected by both pubertal maturation and gonadal steroid milieu. Importantly, both the direction and magnitude of changes in LHRH mRNA that occur with either pubertal maturation or manipulation of gonadal steroids are brain region specific. Puberty is associated with an increase in LHRH mRNA levels, primarily within neurons in the ARC, and LHRH mRNA within the ARC of prepubertal males is strongly regulated by gonadal steroids. In contrast, manipulation of gonadal steroids in adults alters LHRH mRNA levels primarily in cells in the POA. These results indicate that arcuate LHRH neurons play a dominant role in the pubertal increase in LHRH release, and the data are consistent with the hypothesis that the pubertal decrease in responsiveness to steroid negative feedback regulation of LHRH is mediated by a pubertal change in the population of cells in which gonadal steroids most strongly regulate LHRH mRNA.

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.


   Acknowledgments
 
We thank Drs. Julie Bakker and Lydia DonCarlos for constructive comments on early versions of this manuscript.


   Footnotes
 
1 This work was supported by Grant HD-26483, Research Career Development Award HD-00950 (to C.L.S.) and Training Grant NS-07279 (to M.L.K.). Back

2 Present address: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China. Back

3 Present address: Department of Physiology, University of Kentucky, Lexington, Kentucky 40536. Back

Received May 28, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Foster DL 1994 Puberty in the sheep. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, vol 2:411–451
  2. Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, vol 2:363–409
  3. Plant TM 1994 Puberty in primates. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, vol 2:453–485
  4. Terasawa E 1995 Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates. Cell Mol Neurobiol 15:141–164[CrossRef][Medline]
  5. Jakubowski M, Blum M, Roberts JL 1991 Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain. Endocrinology 128:2702–2708[Abstract]
  6. Dutlow CM, Rachman J, Jacobs TW, Millar RP 1992 Prepubertal increases in gonadotropin-releasing hormone mRNA, gonadotropin-releasing hormone precursor, and subsequent maturation of precursor processing in male rats. J Clin Invest 90:2496–2501
  7. Gore AC, Wu TJ, Rosenberg JJ, Roberts JL 1996 Gonadotropin-releasing hormone and NMDA receptor gene expression and colocalization change during puberty in female rats. J Neurosci 16:5281–5289[Abstract/Free Full Text]
  8. Wray S, Hoffman G 1986 A developmental study of the quantitative distribution of LHRH neurons within the central nervous system of postnatal male and female rats. J Comp Neurol 252:522–531[CrossRef][Medline]
  9. Yellon SM, Newman SW 1991 A developmental study of the gonadotropin-releasing hormone neuronal system during sexual maturation in the male Djungarian hamster. Biol Reprod 45:440–446[Abstract]
  10. Gay VL, Plant TM 1987 N-Methyl-D,L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys. Endocrinology 120:2289–2296[Abstract]
  11. Urbanski HF, Ojeda SR 1987 Activation of luteinizing hormone-releasing hormone release advances the onset of female puberty. Neuroendocrinology 46:273–276[Medline]
  12. Plant TM, Gay VL, Marshall GR, Arslan M 1989 Puberty in monkeys is triggered by chemical stimulation of the hypothalamus. Proc Natl Acad Sci USA 86:2506–2510[Abstract/Free Full Text]
  13. MacDonald MC, Wilkinson M 1990 Peripubertal treatment with N-methyl-D-aspartic acid or neonatally with monosodium glutamate accelerates sexual maturation in female rats, an effect reversed by MK-801. Neuroendocrinology 52:143–149[Medline]
  14. Ryan KD, Robinson SL, Tritt SH, Zeleznik AJ 1988 Sexual maturation in the female ferret: circumventing the gonadostat. Endocrinology 122:1201–1207[Abstract]
  15. Sisk CL 1987 Evidence that a decrease in testosterone negative feedback mediates the pubertal increase in luteinizing hormone pulse frequency in male ferrets. Biol Reprod 37:73–81[Abstract]
  16. Sisk CL, Turek FW 1983 Developmental time course of pubertal and photoperiodic changes in testosterone negative feedback on gonadotropin secretion in the golden hamster. Endocrinology 112:1208–1216[Medline]
  17. Barry J, Carette B 1975 Immunofluorescence study of LRF neurons in primates. Cell Tissue Res 164:163–178[Medline]
  18. Silverman AJ, Antunes JL, Abrams GM, Nilaver G, Thau R, Robinson JA, Ferin M, Krey LC 1982 The luteinizing hormone-releasing hormone pathways in rhesus (Macaca mulatta) and pigtailed (Macaca nemestrina) monkeys: new observations on thick, unembedded sections. J Comp Neurol 211:309–317[CrossRef][Medline]
  19. Tang YP, Sisk CL 1992 LHRH in the ferret: pubertal decrease in the number of immunopositive arcuate neurons. Peptides 13:241–247[CrossRef][Medline]
  20. Sisk CL 1990 Photoperiodic regulation of gonadal growth and pulsatile luteinizing hormone secretion in male ferrets. J Biol Rhythms 5:177–186[Abstract/Free Full Text]
  21. Adelman JP, Mason AJ, Haflick JS, Seeburg PH 1986 Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin-release-inhibiting factor in human and rat. Proc Natl Acad Sci USA 83:179–183[Abstract/Free Full Text]
  22. Selmanoff M, Shu C, Petersen SL, Barraclough CA, Zoeller RT 1991 Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic rats. Endocrinology 128:459–466[Abstract]
  23. Wray S, Gainer H 1989 Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions. Mol Endocrinol 3:1197–1206[Abstract]
  24. Zoeller RT, Seeburg PH, Young III WS 1988 In situ hybridization histochemistry for messenger ribonucleic acid (mRNA) encoding gonadotropin-releasing hormone (GnRH): effect of estrogen on cellular levels of GnRH mRNA in female rat brain. Endocrinology 122:2570–2577[Abstract]
  25. Bibeau CE, Tobet SA, Anthony ELP, Carroll RS, Baum MJ, King JC 1991 Vaginocervical stimulation of ferrets induces release of luteinizing hormone-releasing hormone. J Neuroendocrinol 3:29–35
  26. Lambert GM, Rubin BS, Baum MJ 1992 Sex difference in the effect of mating on c-fos expression in luteinizing hormone-releasing hormone neurons of the ferret forebrain. Endocrinology 131:1473–1480[Abstract]
  27. Wersinger SR, Baum MJ 1996 The temporal pattern of mating-induced immediate-early gene product immunoreactivity in LHRH and non-LHRH neurons of the estrous ferret forebrain. J Neuroendocrinol 8:345–359[CrossRef][Medline]
  28. King JC, Rubin BS 1995 Dynamic alterations in luteinizing hormone-releasing hormone (LHRH) neuronal cell bodies and terminals of adult rats. Cell Mol Neurobiol 15:89–106[CrossRef][Medline]
  29. Wiemann JN, Clifton DK, Steiner RA 1989 Pubertal changes in gonadotropin-releasing hormone and proopiomelanocortin gene expression in the brain of the male rat. Endocrinology 124:1760–1767[Abstract]
  30. Toranzo D, Dupont E, Simard J, Labrie C, Coute J, Labrie F, Pelletier G 1989 Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats. Mol Endocrinol 3:1748–1756[Abstract]
  31. Emanuele NV, Jurgens J, La Paglia N, Williams DW, Kelley MR 1996 The effect of castration on steady state levels of luteinizing hormone-releasing hormone (LHRH) mRNA and proLHRH processing: time course study utilizing semi-quantitative reverse transcription/polymerase chain reaction. J Endocrinol 148:509–515[Abstract]
  32. Park Y, Park SD, Cho WK, Kim K 1988 Testosterone stimulates LH-RH-like mRNA level in the rat hypothalamus. Brain Res 451:255–260[CrossRef][Medline]
  33. Rothfeld JM, Hejtmancik JF, Conn PM, Pfaff DW 1987 LHRH messenger RNA in neurons in the intact and castrate male rat forebrain, studied by in situ hybridization. Exp Brain Res 67:113–118[CrossRef][Medline]
  34. Wiemann JN, Clifton DK, Steiner RA 1990 Gonadotropin-releasing hormone messenger ribonucleic acid levels are unaltered with changes in the gonadal hormone milieu of the adult male rat. Endocrinology 127:523–532[Abstract]
  35. Malik KF, Silverman AJ, Morrell JI 1991 Gonadotropin-releasing hormone mRNA in the rat: distribution and neuronal content over the estrous cycle and after castration of males. Anat Rec 231:457–466[CrossRef][Medline]



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