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Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0084
Address all correspondence and requests for reprints to: Kristine Krajnak, Ph.D., Department of Physiology, MS-508 Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0084. E-mail: kmkraj1{at}pop.uky.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous studies have demonstrated that a circadian pacemaker controls the timing of the LH surge (4) and receptive behavior (3), and insures that these rhythms are tightly coupled to the environmental LD cycle. First, treatment with pentobarbital on the morning of proestrus blocks the preovulatory surge of LH. However, a LH surge occurs at the predicted time on the following day, indicating that a daily neurochemical signal is responsible for generating LH surges (5). Second, female rodents housed in LD cycles of varying lengths (i.e. 21–24 h) vary the lengths of their reproductive cycle, such that a single estrous cycle is exactly 4 times the length of the LD cycle to which the animals are exposed (6). Furthermore, animals housed in the absence of any environmental light cues (i.e. constant darkness) continue to show estrous cycles with a period length that is 4 times the length of the endogenous circadian cycle (7). Third, animals that are ovariectomized and treated with estrogen have daily surges of LH that occur at the same time every afternoon (2). Thus, taken together, the studies indicate that the timing of the LH surge and behavioral estrus are regulated by a circadian pacemaker.
In mammals, the endogenous pacemaker that drives virtually all circadian rhythms and entrains these rhythms to the environmental LD cycle is located in the suprachiasmatic nuclei (SCN) of the hypothalamus (8). Evidence indicating that the SCN regulate the timing of the LH surge and receptive behavior comes from several lines of research. Lesions of the SCN abolish estrous cyclicity in gonadally intact females (9, 10), and the daily afternoon surge of LH is eliminated in estrogen-treated ovariectomized females (11). In other studies, females housed in constant darkness and then treated with a phase-shifting agent, triazolam, at a specific time show SCN-dependent shifts in the timing of running wheel activity and concomitant shifts in the timing of the LH surge (12). Finally, changes in the timing of lights on and lights off shift both the timing of locomotor activity and the timing of the LH surge (13). Together, these studies indicate that the circadian pacemaker that couples the timing of the LH surge and receptive behavior to the LD cycle is located in the SCN.
The exact mechanisms by which the SCN synchronize rhythms important for reproduction are unknown. However, numerous studies suggest that vasoactive intestinal polypeptide (VIP), which is synthesized in the ventrolateral portion of the SCN, may be a component of the stimulatory signal that times the LH surge (14, 15, 16, 17). First, studies show that VIP axonal varicosities are apposed to GnRH cell bodies and dendrites (14, 17), and that VIP fibers form synapses directly with GnRH neurons (18). Lesioning the SCN eliminates the VIP input to GnRH neurons (14). Many of the GnRH neurons receiving VIP input express Fos on the afternoon of proestrus, suggesting that VIP may activate GnRH neurons that stimulate the LH surge (17, 19). Finally, central administration of an antibody to VIP (15) or delivery of VIP antisense oligonucleotides to the SCN (16) delays the initiation and attenuates the amplitude of the LH surge. These studies clearly suggest that VIP contributes a stimulatory or permissive timing signal from the SCN to the GnRH neurons. However, a stimulatory role for this neuropeptide is difficult to reconcile with the diurnal rhythm of VIP that exists in males. In males, VIP messenger RNA (mRNA) and protein are at their lowest levels during the day and peak during the night. No studies have been performed in females. Therefore, we examined whether the pattern of VIP expression is different in females and whether the pattern in females depends upon steroidal milieu.
Arginine vasopressin (AVP), a peptide synthesized by neurons in the dorsomedial portion of the SCN, has also been implicated in regulating the timing of the LH surge (20, 21, 22, 23) and receptive behavior (24, 25, 26) in rodents. Anatomical studies show that AVP projections from the SCN synapse on steroid receptor-containing cells in the anteroventral portion of the periventricular nucleus (AVPv) (27, 28). The AVPv is a sexually dimorphic region of the hypothalamus (29, 30) that plays a critical role in generating the LH surge (31). The AVP-containing projection from the SCN to the AVPv is also sexually dimorphic in rats, with the density of the projection being greater in male than in female rats (32). Studies that examined the physiological role of AVP in regulating the LH surge have been contradictory. When AVP is administered intracerebroventricularly (icv), it is inhibitory, delaying the timing and reducing the amplitude of the LH surge (20, 21, 22). However, if AVP is dialyzed directly into the preoptic area, it stimulates LH release (23). AVP also inhibits the expression of sexual receptivity in females (24, 25, 26). Thus, AVP may be another SCN peptide that provides timing cues to GnRH neurons. To test this possibility, we assessed the rhythm in AVP mRNA in the same animals in which we measured VIP.
The overall goal of the present study was to determine whether the diurnal pattern of VIP and AVP gene expression in the SCN differs in female compared with male rats. We hypothesized that because VIP plays a facilitory or permissive role in the proestrous surge of LH, VIP gene expression is increased during the light phase of the LD cycle in females. It was also important to assess whether the pattern of VIP expression in females was modulated by estrogen and specific to the endocrine conditions under which the LH surge occurs. In addition, because AVP may regulate the timing and amplitude of the LH surge, we also examined the rhythmic expression of AVP in females compared with males.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization
Brains were rapidly removed, frozen on dry ice, and stored at
-70 C until they were sectioned (12 µm) in a cryostat. Sections were
thaw mounted onto slides and stored at -70 C until they were processed
for in situ hybridization following a previously established
protocol (33). A total of 52 sections (26 slides) containing the SCN
were collected from each animal. Ten sections (5 slides) containing the
midportion of the SCN (
288–384 µm caudal from rostral border of
the SCN) were chosen for in situ hybridization because the
majority of VIP-containing neurons are located in this region (35, 36),
and AVP is also highly expressed in this part of the SCN (37). Of the 5
slides chosen for each animal, 3 slides (every other slide) were
processed for VIP, and the other 2 slides were processed for AVP
in situ hybridization. A riboprobe for VIP was generated
using a 500-bp human VIP complementary DNA (cDNA) directed against
exons 3–6 of the VIP/peptide histidine isoleucine gene
(provided by Dr. R. H. Goodman, Vollum Institute, Portland,
OR). For AVP, a riboprobe was generated using a 241-bp rat cDNA
directed against exon C of the AVP gene (provided by Dr. T. Sherman,
Georgetown University, Washington DC). Both riboprobes were transcribed
using 50 µM total UTP (VIP: 12.5 µM
[35S]UTP and 37.5 µM unlabeled UTP and SP6
polymerase; AVP: 37.5 µM [35S]UTP with 12.5
µM unlabeled UTP and SP6 polymerase). Slides were thawed,
fixed with 4% paraformaldehyde, and dehydrated using a series of
increasing concentrations of ethanol. Hybridization buffer (50 µl)
containing 400 ng/ml labeled VIP complementary RNA (cRNA) or 200 ng/ml
AVP cRNA was applied to each slide. In preliminary studies, saturation
curves were generated and revealed that these concentrations of cRNA
produced maximal labeling without significantly increasing background
labeling. Slides were incubated in humid chambers at 55 C for
18 h, then washed under stringent conditions, dehydrated with
ethanol, coated with Kodak NTB2 emulsion (diluted 1:1 with distilled
water; Eastman Kodak, Rochester, NY) and stored at 4 C. Slides
processed for AVP were developed 5 days after emulsion coating, and
slides processed for VIP were developed 10 days after emulsion coating.
All slides were counterstained with 0.05% toluidine blue so that
individual cell bodies could be identified.
All slides were examined for the presence of labeling in the SCN. If
the SCN from an individual animal was damaged, mRNA levels were not
quantified in those slides. Therefore, in some animals, AVP was not
quantified, and in others, VIP was not quantified. Gene expression was
quantified using the Bioquant OS/2 Image Analysis System (R&M
Biometrics, Nashville, TN). Cells were imaged under brightfield
microscopy at a total magnification of x400. At this magnification,
the perimeter of each cell was outlined so that the area of the cell
covered by grains could be measured. All cells in the SCN that were
covered by grains were analyzed. Lighting and contrast levels were
standardized before taking measurements to assure that all slides were
assessed under the same conditions. Background was assessed by taking
measurements over unlabeled cells outside the area of interest. Cells
with a value 5 times higher than background were considered labeled.
VIP labeling was seen over cell bodies located primarily in the
ventrolateral portion of the SCN, and AVP labeling was seen over cell
bodies located in the dorsomedial SCN (Fig. 1
, A and B).
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Statistical analyses
VIP and AVP mRNA levels per cell and the number of VIP or AVP
mRNA-expressing cells per section in the SCN of ovx,
ovx-E2, and proestrous females were analyzed using ANOVA
(three treatments x five times of day) to determine whether gene
expression showed a diurnal rhythm and whether this rhythm was
influenced by ovarian hormones. Serum estradiol levels in ovx females
were below the lower limit of detectability of the assay. Thus,
estradiol levels in proestrous and ovx-E2 females were
analyzed using ANOVA (two treatment x five times of day). One-way
ANOVA was used to analyze the effects of time on VIP and AVP gene
expression in males. For all ANOVAs, significant interactions were
further analyzed using one-way ANOVAs, and post-hoc pairwise
comparisons were made using the Student-Newman-Keuls test.
P < 0.05 was considered significant for all tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Analysis of VIP
mRNA levels in the SCN of females revealed a significant main effect of
treatment [F(2, 75) = 3.82; P < 0.03] and of time
[F(4, 75) = 2.89; P < 0.03], but the interaction
between treatment and time of day was not significant, indicating that
although hormonal status alters the average level of VIP mRNA, it does
not significantly alter the diurnal pattern of VIP gene expression in
the SCN of females. Pairwise comparisons of the effects of treatment
demonstrated that overall, VIP mRNA levels were higher in ovx females
than in proestrous or ovx-E2 females (Fig. 3; P < 0.05). Further
analysis of the main effect of time using the Student-Newman-Keuls test
showed that in females, VIP gene expression in the SCN was lower at
0300 h than at any other time of day (Fig. 4; P < 0 0.05). The
number of VIP-labeled cells per section did not change with time or
treatment (Fig. 4). Thus, although estrogen does not significantly
alter the diurnal pattern of VIP expression in the SCN, it does affect
average VIP mRNA levels.
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; P < 0.05). This is
different from the pattern seen in females, in which VIP levels were
lowest at 0300 h and increased after lights on. However, as in
females, this rhythm was due to changes in mRNA levels per cell, as
time of day did not affect the number of cells labeled for VIP in the
SCN of males (Fig. 4).
Analysis of AVP mRNA levels in the SCN of female rats revealed that
there was a significant main effect of time [F(4, 83) = 26.86;
P < 0.001], but no effect of treatment and no
interaction between time and treatment (Fig. 5). Pairwise comparisons made using
Student-Newman-Keuls tests revealed that AVP gene expression was lowest
at 0300 h and then significantly increased between 0300–0800 h
and again between 0800–1600 h (Fig. 6;
P < 0.05). Between 1600–2300 h, AVP mRNA levels
significantly declined (P < 0.05). Analysis of the
number of AVP mRNA-expressing cells per section also showed a main
effect of time [Fig. 6; F(4, 83) = 4.62; P < 0.05],
but as with measurements of AVP mRNA per cell, there was no effect of
treatment and no interaction. The effect of time of day on cell number
was similar to that observed in terms of AVP mRNA per cell, with cell
number per section being lowest at 0300 h and then gradually
increasing to peak levels between 1200–1600 h (P <
0.05). The number of cells per section labeled for AVP then declined
slightly between 1600–2300 h.
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; P < 0.05). The AVP
mRNA level then remained stable between 1200–1600 h, but declined
between 1600–2300 h (P < 0.05). There was also a
significant effect of time of the number of AVP-expressing cells in the
SCN of males [F(4, 17) = 3.31; P < 0.05]. Again,
this effect was identical to that seen in females, with the number of
AVP mRNA-expressing cells being lowest at 0300 h and increasing to
peak levels between 1200–1600 h (P < 0.05). The
number of AVP-expressing cells significantly decreased between
1600–2300 h (Fig. 6).
Serum estradiol levels in ovx females were below the level of
detectability of the assay (10 pg/ml) and therefore were not included
in the analyses. Circulating estradiol levels in proestrous and
ovx-E2 females are listed in Table 1. There was an interaction between
treatment and time of day on serum estradiol levels [by two-way ANOVA:
F(4, 55) = 3.19; P < 0.03]. Further analysis
demonstrated that in proestrous females, estradiol levels increased
over the day [F(4, 31) = 5.64; P < 0. 03] and were
higher at 1600 h than at any other time during the day
(P < 0.05). In ovx-E2 females, circulating
estradiol levels did not significantly fluctuate over the day.
Estradiol concentrations in E2-treated females were not
significantly different than concentrations in proestrous females at
any time of the day.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple studies have shown that VIP may convey circadian information from the SCN to GnRH neurons, and that VIP may coordinate the timing and facilitate the generation of the LH surge (15, 16, 17, 19). Based on these studies, we hypothesized that VIP mRNA would be high during the day, before or during the time of the LH surge. Our data support this hypothesis; VIP mRNA levels in the SCN of female rats are lowest at 0300 h, 1 h before lights on, and increase during the light phase of the cycle. The presence of similar patterns of VIP expression in all groups of females leads us to conclude that the pattern of expression of VIP is a neurochemical component that may be necessary to time the LH surge, but is not sufficient to induce the surge in the absence of estrogen. Although VIP release and peptide concentrations have not been measured in the SCN of females, work in males suggests that changes in VIP mRNA levels are reflected by changes in peptide content, and that mRNA and peptide content within the SCN reach peak levels at approximately the same time of day (38). Thus, it is likely that VIP mRNA and peptide content are also synchronized in females, and that fluctuations in mRNA reflect concurrent fluctuations in peptide content.
The pattern of VIP gene expression was not significantly altered by ovarian hormones. However, the average levels of VIP mRNA were. Overall, VIP mRNA was higher in ovx than in proestrous or ovx-E2 treated females. These results are inconsistent with a previous study that suggested that VIP mRNA was increased in gonadally intact or ovx-E2 treated animals compared with that in ovx animals (39). Several differences in the two studies may account for these discrepancies. First, our study examined VIP mRNA levels within SCN neurons of individual animals. Gozes et al. (39) pooled dissected hypothalamic tissue and assessed RNA using hybridization blot analysis. Although the majority of VIP synthesized within the hypothalamus is produced in the SCN, neurons in the paraventicular and periventricular nuclei also synthesize VIP (40). Thus, it is possible that the increase in VIP in response to estrogen seen in the previous study was due to changes in one of these other regions. The second major difference in these two studies was that our animals were ovx for 7 days, and animals in the Gozes et al (39) study were ovx for 3 weeks to 2 months. Although serum estradiol levels in our ovx animals were below the limit of detectability of the assay 1 week after ovariectomy, it is possible that the absence of estrogen for extended periods, such as in the Gozes et al. (39) study, results in a decrease in VIP mRNA, whereas shorter term ovariectomy results in slight increases in VIP.
The pattern of VIP gene expression we observed in males is consistent with the results of previous studies showing that VIP mRNA (36, 41, 42, 43) and peptide concentrations in the SCN (44, 45) decrease after lights on. The finding that VIP mRNA levels in the SCN of males are lower than those in females during the light phase of the cycle is also consistent with work showing that hypothalamic peptide concentrations are lower in males than in females during the middle of the day (46). We did not assess the effects of castration on VIP levels in males in this study. However, previous studies indicate that neither castration (39, 47) nor estrogen or testosterone replacement (39) alters VIP peptide or mRNA levels in the hypothalamus of male rats. Thus, we conclude that the sex difference in the rhythmic expression of VIP mRNA in rats is not due to the activational effects of gonadal steroids.
This and other studies suggest that the sex-related difference in the rhythm of VIP mRNA may result from the organizational effects of steroids on the SCN and/or on its afferents and efferents during perinatal development. These profound organizational effects of steroids influence numerous brain regions involved in the regulation of reproduction. It appears that the SCN become sexually dimorphic during the perinatal period under the influence of steroids (3, 48), and the ability of ovarian steroids to alter activity rhythms in adult females depends upon the perinatal organization of the circadian pacemaker. In adult female rodents, gonadal steroid fluctuations over the estrous cycle or steroid treatments alter both the length of the animals free running activity rhythm and the relationship of that rhythm to the LD cycle (49, 50, 51, 52). In contrast, males and females that were androgenized perinatally have shorter circadian periods and do not show consistent changes in activity rhythms in response to estradiol treatment (53). This indicates that the SCN are sexually differentiated during development and that this differentiation determines the response of the SCN to circulating steroids later in development. Previous studies also suggest that sexual differentiation of the SCN during development may impact the rhythmic pattern of VIP expression in the SCN (54) and hypothalamic VIP concentrations (55).
The rhythm of AVP gene expression in male and female rats is identical, and AVP mRNA levels were not altered by steroid treatment in females. Our results are consistent with previous work describing the rhythmic expression of AVP mRNA in the SCN of male rats (56). We had predicted that because AVP may play a role in regulating the timing and the amplitude of the LH surge (20, 21, 22, 23), the rhythmic expression of this peptide may be different in males and females. Our results do not confirm this prediction. Although AVP mRNA is high during the afternoon of proestrus, data collected from males suggests that AVP release (57) and peptide content (45) within the SCN are out of phase with mRNA, with peptide release and content reaching peak levels shortly after lights on and declining during the afternoon. Thus, AVP concentrations in the SCN are probably low during the LH surge. Our results are more consistent with the hypothesis that the decline in AVP provides a stimulatory signal for the LH surge (20, 21, 22). However, we cannot rule out the possibility that the subset of AVP neurons projecting to the preoptic area increase their activity before the surge, thereby stimulating the release of GnRH and LH (23).
The decline in AVP seen during the late day and after lights off may be important for timing sexual receptivity in females. As icv administration of an AVP antagonist while AVP is high in the SCN facilitates sexual receptivity, and icv administration of AVP while AVP levels in the SCN are low inhibits sexual receptivity (26), the decline in AVP seen during the late afternoon and evening may be important for allowing the timed expression of receptive behavior in female rats (24, 25, 26).
The failure to find a difference in the rhythmic expression of AVP mRNA in the SCN does not mean that this system is not sexually differentiated. As mentioned previously, AVP projections from the SCN to the AVPv are more dense in males than in females (32). Thus, not only is it the rhythmic expression of these peptides that is important, but the efferent connections these cells make may also determine how they regulate the overt expression of rhythms in hormone release and behavior. To fully understand the mechanisms responsible for the differences in rhythmic behaviors in males and females, more detailed anatomical studies describing the projections of these two peptide systems will need to be performed.
In summary, the rhythmic expression of VIP in the SCN is different in males and females. The high levels of VIP mRNA seen in the SCN of the female during the day support the suggestion that VIP may serve as a stimulatory signal regulating the timing of the LH surge. In contrast, the rhythmic expressions of AVP mRNA in the SCN of males and females are identical. This suggests that the sexually differentiated functions of AVP may be regulated via specific projections made by these neurons rather than by the pattern of peptide synthesis and release.
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Acknowledgments |
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Footnotes |
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Received March 6, 1998.
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