- Email: [email protected]
Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat
Molecular and Cellular Endocrinology 252 (2006) 120–125
Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat Tamar D. Schirman-Hildesheim, Nurit Ben-Aroya, Yitzhak Koch ∗ Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
Abstract We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9 h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: GnRH mRNA; GnRH receptor mRNA; Estrus cycle; Ovariectomized rats; Daily expression pattern; Rat hypothalamus; Rat pituitary
1. Introduction Reproductive functions in all vertebrates are orchestrated by the hypothalamic gonadotropin-releasing hormone, GnRH. This decapeptide is released from hypothalamic nerve endings into the portal system, leading it into the anterior pituitary, where it induces the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. GnRH and its receptor (GnRH-R) are also produced in a variety of extra-hypothalamic organs and glands, where their role has not been elucidated yet. We have recently described tissue-specific fluctuations in the levels of GnRH and GnRH-R mRNA in the hypothalamus, pituitary and ovary throughout the estrus cycle (Schirman-Hildesheim et al., 2005). Subsequently, we wished to discriminate between estrus cycle stage-dependant regulation by ovarian factors and underlying circadian oscillations in GnRH and GnRH-R expression.
∗
Corresponding author. Tel.: +972 8 9342790; fax: +972 8 9344131. E-mail address: [email protected] (Y. Koch).
0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.03.010
The circadian system plays a central role in mammalian reproduction (Goldman, 1999; Barbacka-Surowiak et al., 2003; Kennaway, 2005). In many spontaneously ovulating species, including rodents, ovulation and the preceding gonadotropin surge occur at roughly the same time in every cycle. Moreover, a shift in the timing of light onset leads to a corresponding shift in the timing of ovulation (Hoffmann, 1969). In a time-free environment (e.g. continuous dim light illumination) the estrus cycle maintains its circadian rhythmicity (Goldman, 1999). A daily neurogenic stimulus for luteinizing hormone (LH) surges has been demonstrated in rats by Everett and Sawyer (1949). The same researchers later showed that blocking of this neurogenic stimulus, using pentobarbital, delays the LH surge and ovulation by precisely 24 h (Everett and Sawyer, 1950). Later studies (Legan et al., 1975), using ovariectomized (OVX) rats, have pointed at the estrogen-dependency of these daily evening – centrally regulated – LH surges. In fact, steroid hormones have since been shown to play an important role in fine tuning the circadian clock (Abizaid et al., 2004, 2005; Sellix et al., 2004). Thus, mammalian reproduction is regulated by circadian mechanisms that are entrained to the light:dark cycles and are under the influence of ovarian factors.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
Central and peripheral circadian rhythms are classically thought to be regulated by the suprachaismatic nucleus (SCN) of the hypothalamus, which is itself entrained to the pattern of daylight. The SCN has been shown to be anatomically connected to GnRH-producing neurons in the hypothalamus (Van Der Beek et al., 1997). Recent evidence however, supports the contention that peripheral tissues and sites outside of the SCN, including GnRH-producing neurons (Gillespie et al., 2003), are capable of self-sustaining circadian rhythms even in the absence of central coordination (Yoo et al., 2004). Furthermore, since melatonin receptors are present in various tissues outside the brain, such as the ovary (Soares et al., 2003) and pituitary (Williams, 1989), synchronization of circadian rhythms with daylight could be achieved locally. In the present study, we searched for daily, ovarianindependent, variations in the levels of GnRH and GnRH-R mRNA, which might underlie the tissue-specific patterns of expression observed throughout the estrus cycle. To this end, we abolished the regulatory influence of ovarian factors by ovariectomizing adult rats. We then measured levels of pituitary and hypothalamic GnRH and GnRH-R expression during different times of day. Indeed, we found that GnRH and GnRH-R mRNA concentrations fluctuate during the day, in a tissue-specific and ovarian-independent manner. Moreover, we extended our previous study of estrus cycle patterns in GnRH and GnRH-R expression by studying additional time points during diestrus-I, -II and estrus, so as to present a comparison of daily expression patterns in intact versus OVX rats. 2. Materials and methods 2.1. Animals All animals were purchased from Harlan Laboratories (Rehovot, Israel) and all experiments were carried out in compliance with the regulations of the Weizmann Institute of Science and using accepted standards of humane animal care. Seven to nine weeks old female Wistar rats were used. Animals were housed under constant conditions of temperature and humidity with lights on between 6 a.m. and 8 p.m. and food available ad libitum. For the intact rat study, estrus cycles were monitored via vaginal smears and only rats showing at least three consecutive 4-day cycles were used. Animals were sacrificed by decapitation at the times listed in Table 1. For the OVX rat study, 7–8-week old Wistar rats were ovariectomized under anesthesia (Ketamine HCl and diazepam, 7.5 and 0.375 mg per 150 g animal, respectively). One month later the animals were sacrificed by decapitation at the times listed in Table 1. After decapitation, tissues were immediately removed and placed in 10 volumes of RNA Later (Ambion, Austin, TX, USA) until subsequent RNA extraction. The hypothalamus was dissected out to a depth of ∼3 mm with the following borders: the anterior edge of the optic chiasm, the anterior edge of the mammillary bodies and the two hypothalamic sulci on either lateral side. Both ovaries were removed and oviducts were examined for the presence of ova only in the groups sacrificed in the morning of estrus. OVX rats were examined to ascertain the absence of ovary fragments. The anterior pituitary was removed together with the attached posterior lobe.
121
Table 1 Times of animal sacrifice and sample sizes Time of day 06:30 07:30 09:00 10:00 12:00 14:00 15:00 16:00 17:00 19:00 20:00 21:30 22:00
Diestrus-I (n) Diestrus-II (n) Proestrus (n) Estrus (n) OVX (n)
4 12 5
10 6
4
4
6
7
10 4 4 5 8 10
7
6 6 8
5
5 5
5 5
7 5 5 5
6 6
ethanol and loaded onto an RNEasy mini-column (Qiagen GmbH, Hilden, Germany). The procedures for RNA isolation and purification, as well as on-column DNAse treatment (Qiagen), were then carried out as detailed in the manufacturer’s instructions. RNA samples were eluted in nuclease-free water (Qiagen). RNA concentration was quantified using a NanoDrop machine (NanoDrop Technologies, Wilmington, Delaware, USA) and its RNA purity was assessed on the same machine using the 260:280 and 260:230 nM ratios. All samples had 260:280 nM ratios between 1.8 and 2.1, and 260:230 nM ratios above 1.7. RNA integrity was assessed by examining the 28S and 18S bands of representative samples loaded onto a 1.5% agarose gel stained with ethidium bromide.
2.3. Reverse transcription (RT) For each tissue, equal amounts of all RNA samples were reversetranscribed simultaneously. Hypothalamic RNA samples (2 g each) were reverse-transcribed using M-MLV RNase H+ (Promega, Madison, WI, USA) according to the manufacturers’ instruction. Each reaction contained: 0.5 g Oligo-dT (Amersham Biosciences, Piscataway, NJ, USA), 0.52 mM of each dNTP (MBI Fermentas, St. Leon-Rot, Germany), 25 units RNAguard RNase inhibitor (Amersham Biosciences), 5 l of the 5× M-MLV reaction buffer (Promega) and 200 units of the enzyme in a total volume of 25 l. Pituitary RNA samples (4 g each) were reverse-transcribed using SuperScript II RNase H− Reverse Transcriptase kit (Invitrogen Life Technologies, Carlsbad, CA, USA). Each 20 l reaction contained: 0.5 g Oligo-dT (Amersham Biosciences), 0.5 mM of each dNTP (MBI Fermentas, Germany), 40 units porcine liver ribonuclease-inhibitor (Takara Bio Inc., Shiga, Japan), 2 l 0.1 M DTT, 4 l of the 5× first-strand buffer (Invitrogen) and 40 units of the enzyme. All RT reactions were performed at 42C and contained a negative control, which consisted of nuclease-free water instead of RNA. The linearity of the RT reaction was evaluated as described previously (Schirman-Hildesheim et al., 2005).
2.4. Gene-specific primers and taqman hybridization probes Primers were designed on two different exons so as to span one intronic sequence. TaqMan hybridization probes were designed to span an exon–exon junction (TIB-Molbiol, Berlin, Germany). All primer and probe sequences, PCR product sizes, product identity and annealing temperatures have been reported previously (Schirman-Hildesheim et al., 2005).
2.5. Relative real-time PCR 2.2. RNA purification Tissues were removed from the RNA later, weighed and homogenized in 0.5 mL Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). Following the addition of 100 l chloroform and phase separation by centrifugation (at 4C), the aqueous layer was washed with an equal volume of 70%
All real-time PCR reactions were carried out exactly as detailed elsewhere (Schirman-Hildesheim et al., 2005). Each real-time PCR reaction included a no-template control (NTC) as well as five or six serial four-fold dilutions, in duplicates, of a cDNA pool containing all experimental samples of the respective tissue. The pre-normalized DNA quantity of each gene in every sample was
122
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
estimated relative to this dilution series. This dilution series also served to assess the reaction performance (E and R2 ). The threshold cycle (Ct) was set so as to obtain the highest reaction efficiency and correlation coefficient. For normalization of gene expression, a panel of four candidate reference genes (HPRT, Cyclophillin, RPL19 and -actin) were tested in all experimental samples in order to identify the most stably expressed genes in all tested times in both intact and OVX rats. Primer sequences and reaction conditions for all tested reference genes, as well as the criteria used to select the most stable reference genes, have been described previously (ibidem). Since not all samples of a particular tissue or condition (intact versus OVX) could be assayed simultaneously for each gene, a common set of at least five ‘calibrator’ samples was included in every reaction for inter-reaction calibration (see below). Additionally, in order to compare between expression levels in OVX rats versus intact rats, each qPCR reaction with OVX samples also contained a set of samples from the same tissue of intact animals sacrificed at 10 h of diestrus-I. The latter samples served to calibrate the relative mRNA levels in OVX rats to those in intact rats (see below).
have been normalized to that at 10 h of diestrus-I, as explained in Section 2 (Section 2.6). It is important to note however, that in order to quantitatively compare the relative mRNA amounts in intact versus OVX rats, one must assume that the reference genes’ mRNA, used for normalization, are equally abundant in both conditions. 3.2. Relative daily levels of GnRH expression in intact and OVX rats
3. Results
Significant daily fluctuations in the relative expression levels of GnRH were found in intact as well as OVX rats in both the hypothalamus and pituitary (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.05). In the hypothalamus of intact rats (Fig. 1A, top graph), non-significant variations were observed during diestrus-I and estrus, though evening levels seem higher on both days, as compared to morning levels. During diestrusII and proestrus, significantly high levels were found in the evening, at 19 and 17 h, respectively. In the hypothalamus of OVX rats (Fig. 1A, bottom graph), low mRNA levels were detected at 14, 17, 19 and 22 h, whereas peak levels were recorded at 12, 16 and 20 h. Overall, similar relative amounts of GnRH mRNA were measured in intact and OVX rats, if one assumes equal abundance of RPL19 and Cyclophillin in both conditions. In the pituitary of intact rats (Fig. 1B, top graph), GnRH mRNA expression remained rather stable on diestrus-I. Significant fluctuations occurred during diestrus-II and proestrus. On the former, peak levels were measured at noon, with low levels in the afternoon. On proestrus, GnRH mRNA was at a lull in the morning and fluctuated thereafter. GnRH levels were highest throughout the estrus day as compared to all other times of the cycle (except for noon of diestrus-II), and remained stable during this day. In the pituitary of OVX rats (Fig. 1B, bottom graph) we noted a rather different daily GnRH expression pattern, with a stark rise at 9 h (eight-fold increase) followed by much lower levels (estrus cycle levels) throughout the remainder of the day.
3.1. Stably expressed reference genes in the pituitary and hypothalamus of intact and OVX rats
3.3. Relative daily levels of GnRH-R expression in intact and OVX rats
In our hands, Cyclophillin and RPL19 were the most stably expressed genes across all hypothalamic and pituitary samples of intact rats as well as in hypothalamic samples of OVX rats. Cyclophillin and -actin were found to be the most stably expressed reference genes among pituitary samples of OVX rats. Details regarding the selection criteria used have been described elsewhere (Schirman-Hildesheim et al., 2005). The two most stably expressed reference genes were used to normalize the expression of GnRH and GnRH-R in each sample, as described in Section 2. Mean relative expression values (±standard error of the mean, S.E.M.) in each time group in both experimental conditions (OVX versus intact) are plotted in Fig. 1. The daily expression patterns in OVX animals are quadruplicated in the graphs, so as to cover a range of 4 days, enabling an easier comparison to each day of the estrus cycle. All expression levels
Statistical analysis of hypothalamic GnRH-R expression pattern across the estrus cycle (Fig. 1C, top graph) reveals only non-significant variations, possibly due to relatively high interindividual variability (ANOVA, p > 0.05). Nonetheless, significant local variations can be noted: (i) higher levels at 16 h versus 12 h on diestrus-I, (ii) higher levels at 12 h versus 10 h on diestrus-II (Student’s t-test, p < 0.05). Moreover, on proestrus, GnRH-R mRNA peaked at 14 and 21 h. On the day of estrus, GnRH-R mRNA seemed to remain constant. In the hypothalamus of OVX rats (Fig. 1C, bottom graph), comparison of each pair of time points using Student’s t-test resulted in significant differences at 16 h as compared to 9 or 17 h. Overall, GnRH-R mRNA concentration was slightly higher in OVX rats as compared to intact rats, assuming equal abundance of the reference genes.
2.6. Data analysis and statistics The relative amounts of GnRH or GnRH-R mRNA were calculated in the following manner: (i) the pre-normalized DNA quantity in each sample (obtained from the dilution series) was divided by the geometric mean of the DNA quantities of the calibrator samples (see above) to account for inter-reaction differences; (ii) this calibrated DNA quantity in each sample was then divided by the geometric mean of the DNA quantities of the two most suitable reference genes (Vandesompele et al., 2002), to produce a ‘normalized DNA quantity’; (iii) the mean normalized DNA quantity was computed for each time group, in every tissue; (iv) finally, every mean normalized DNA quantity was normalized to that of the diestrus-I 10 h group (assayed in the same qPCR run, as described above) to obtain comparable results from intact and OVX samples. The final values obtained are hereafter referred to as the ‘relative expression levels’ of GnRH or GnRH-R. All statistical analyses were performed using JMP IN Statistical Discovery Software, Version 5.1 (SAS Institute Inc., Cary, NC, USA). For each tissue and gene the distribution of the obtained relative amounts of GnRH and GnRH-R mRNA in all samples were tested for normality using the Kolmogorov–Smirnov–Lilliefor’s test. Statistical evaluation of differences between time groups was performed using both parametric and non-parametric multiple comparison tests (one-way ANOVA, and the Wilcoxon rank-sum test, respectively), followed by pairwise comparisons of means using the least significant difference (LSD) test and the Student’s t-test at confidence level of 95%.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
123
Fig. 1. Daily expression patterns of GnRH and GnRH-R mRNA in the hypothalamus and pituitary of intact and ovariectomized rats. GnRH expression in the hypothalamus of intact (A, top panel) and OVX rats (A, bottom panel); GnRH expression in the pituitary of intact (B, top panel) and OVX rats (B, bottom panel); GnRH-R expression in the hypothalamus of intact (C, top panel) and OVX rats (C bottom panel); GnRH-R expression in the pituitary of intact (D, top panel) and OVX rats (D, bottom panel). Numbers on the abscissa represent hours of the day. The days of the estrus cycle is indicated above the top panel in each graph. The daily expression patterns detected in OVX animals are quadruplicated in the graphs, so as to cover a range of 4 days, enabling an easier comparison to each day of the estrus cycle. Values are mean mRNA amounts ± S.E.M. (arbitrary values) of 4–12 rats per time group, as indicated in Table 1. Relative mRNA expression levels were calculated as described in Section 2 (Sections 2.5–2.6). Different letters indicate statistically significant differences between groups (Student’s t-test, p < 0.05).
In the pituitary, we observed significant variations in the level of GnRH-R mRNA across the estrus cycle (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.0001; Fig. 1D, top graph) as well as on each day except for estrus, when they were low but stable. Thus, on diestrus-I, GnRH-R mRNA was most abundant at 10 h, lowest at 19 h, with a lower peak at 14 h. A rather similar pattern was observed in the pituitary of OVX rats (Fig. 1D,
bottom graph), where high levels occurred at 9 h, and low levels at 19 h, with an intermittent peak at 14 h (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.05). In OVX rats, GnRH-R was also high at 22 h. We did not sample this time in intact rats. On diestrus-II, GnRH-R was expressed at highest levels in the evening, at 19 h, which was also its estrus cycle peak time. On proestrus, pituitary GnRH-R expression was low in the morn-
124
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
ing and fluctuated during the remainder of the day, much like its ligand. It appears that, generally, GnRH-R mRNA is more abundant in OVX versus intact rats (3–12-fold difference), assuming equal reference genes abundance. 4. Discussion To the best of our knowledge, this is the first demonstration of a GnRH and GnRH-R mRNA expression pattern in the pituitary and hypothalamus of castrated animals. Snabes et al. (1977) have previously demonstrated daily changes in hypothalamic GnRH content in untreated OVX rats, such that higher concentrations were observed in the morning versus the afternoon. The mRNA expression pattern we found is quite different and involves fluctuations throughout the day, with a peak almost every 4 h (Fig. 1A, bottom). Interestingly, we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred slightly earlier, at 17 h (Fig. 1A, top). Similarly, in OVX animals GnRH expression was also found to be higher in the evening, at 20 h, slightly later than in intact animals. It is possible that ovarian factors, such as estrogen, serve to advance the circadian clock governing hypothalamic GnRH production. In support of this hypothesis, estrogen has recently been shown to modulate hypothalamic expression of transcription factors, which precede the expression of clock gene expression in the SCN (Abizaid et al., 2004). During the estrus cycle, we noted an interesting – though weak – hypothalamic GnRH-R expression pattern, consisting of higher levels in the afternoon of each day except on estrus (Fig. 1C, top). Thus, GnRH-R rose at 16 h on diestrus-I, paralleling a peak in OVX rats (Fig. 1C, bottom), and at 12 and 14 h of diestrus-II and proestrus, respectively. This pattern should further be confirmed and its physiological significance is to be unraveled. It is noteworthy however, that the rhythmic expression of hypothalamic GnRH and GnRH-R does not necessarily depend on innervations from the SCN since clock genes have been reported to be expressed within GnRH-producing neurons themselves, in vitro (Gillespie et al., 2003). The daily GnRH expression pattern we observed in the pituitary of OVX rats is rather striking, with a clear peak at 9 h (Fig. 1B, bottom). This pattern is not paralleled on any day of the estrus cycle (Fig. 1B, top). As we mentioned earlier, high hypothalamic GnRH concentrations in OVX rats have previously been recorded in the morning (Snabes et al., 1977). One could tentatively speculate that the higher morning hypothalamic GnRH content is accompanied by higher release into the portal system, which might positively feedback upon the pituitary GnRH-synthesizing cells. An alternative possibility stems from the recent demonstration that pituitary cells are capable of self-sustained circadian oscillations for several days in isolation (Yoo et al., 2004). Thus, GnRH expression in pituitary cells could be governed by local oscillatory molecular mechanisms with a circadian rhythm. The latter could be entrained to daylight via melatonin signaling (Williams, 1989). Pituitary GnRH-R mRNA levels in OVX rats also seem to be higher in the morning, at 9 h (Fig. 1D, bottom). Unlike GnRH,
high levels of GnRH-R expression were also recorded at 14 and 22 h. Though we did not sample intact animals at 22 h on each day of the estrus cycle, the expression pattern obtained on diestrus-I resembles that of OVX rats. On other days of the cycle, the daily expression pattern is quite different (Fig. 1D, top). The physiological significance of these observations is not clear. Interestingly however, GnRH-R mRNA concentrations seem, overall, higher in OVX than in intact rats, as previously demonstrated (Kakar et al., 1994). A similar phenomenon has also been demonstrated at the protein level (Liscovitch et al., 1984). We provide here a more elaborate outline of hypothalamic and pituitary GnRH and GnRH-R expression throughout the estrus cycle, to supplement our previous report (Schirman-Hildesheim et al., 2005). Of note are the following new observations, which we cannot at this time correlate with circadian features seen in OVX rats: (i) high concentrations of pituitary GnRH mRNA throughout estrus, relative to all other days in the cycle (except 14 h of diestrus-II; Fig. 1B, top); (ii) a sharp increase in pituitary GnRH-R production in the evening of diestrus-II, which probably serves to prepare, prime, the gonadotropes for the following day’s hormonal surge (Fig. 1D, top). These observations are to be further confirmed and interpreted in terms of biological significance. We have previously described tissue-specific estrus cycle expression patterns of GnRH and its receptor, and suggested that these genes are locally regulated in accordance with the animal’s reproductive state, or phase of the estrus cycle (Schirman-Hildesheim et al., 2005). Moreover, we speculated that GnRH might play a role in the preparation of the pituitary and ovary for the imminent preovulatory surge, possibly via local GnRH-gonadotropin axes. The contribution of underlying circadian fluctuations in GnRH and GnRH-R expression, which emerge in the absence of ovarian factors, to the estrus cycle patterns of these genes’ expression should be further investigated. It would be interesting to verify the dependence of the observed circadian expression patterns on the lighting periodicity and timing. Indeed, ‘true’ circadian rhythms display not only a circa-24 h periodicity, which is maintained in a time-free environment (e.g. constant dim light), but also entrainment to environmental light–dark cycles (MooreEde et al., 1982; Sellix et al., 2004). The evolutionary advantage of synchronizing reproductive functions to day time, such that ovulation and receptivity concur with the animal’s potential social interaction, is a well known phenomenon, which has been reviewed at length (Van Der Beek, 1996; Barbacka-Surowiak et al., 2003; Goldman, 1999). Acknowledgement This study was supported by the Nella & Leon Benoziyo Center for the Neurosciences and the German-Israeli Foundation for Scientific Research and Development (GIF). References Abizaid, A., Mezei, G., Horvath, T.L., 2004. Estradiol enhances light-induced expression of transcription factors in the Scn. Brain Res. 1010, 35–44.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125 Abizaid, A., Mezei, G., Thanarajasingam, G., Horvath, T.L., 2005. Estrogen enhances light-induced activation of dorsal raphe serotonergic neurons. Eur. J. Neurosci. 21, 1536–1546. Barbacka-Surowiak, G., Surowiak, J., Stoklosowa, S., 2003. The involvement of suprachiasmatic nuclei in the regulation of estrous cycles in rodents. Reprod. Biol. 3, 99–129. Everett, J.W., Sawyer, C.H., 1949. A neural timing factor in the mechanism by which progesterone advances ovulation in the cyclic rat. Endocrinology 45, 581–595, llust. Everett, J.W., Sawyer, C.H., 1950. A 24-hour periodicity in the “Lh-release apparatus” of female rats, disclosed by barbiturate sedation. Endocrinology 47, 198–218. Gillespie, J.M., Chan, B.P., Roy, D., Cai, F., Belsham, D.D., 2003. Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting Gt1-7 neurons. Endocrinology 144, 5285–5292. Goldman, B.D., 1999. The circadian timing system and reproduction in mammals. Steroids 64, 679–685. Hoffmann, J.C., 1969. Light and reproduction in the rat: effect of lighting schedule on ovulation blockade. Biol. Reprod. 1, 185–188. Kakar, S.S., Grantham, K., Musgrove, L.C., Devor, D., Sellers, J.C., Neill, J.D., 1994. Rat gonadotropin-releasing hormone (Gnrh) receptor: tissue expression and hormonal regulation of its Mrna. Mol. Cell Endocrinol. 101, 151–157. Kennaway, D.J., 2005. The role of circadian rhythmicity in reproduction. Hum. Reprod. Update 11, 91–101. Legan, S.J., Coon, G.A., Karsch, F.J., 1975. Role of estrogen as initiator of daily Lh surges in the ovariectomized rat. Endocrinology 96, 50–56. Liscovitch, M., Ben-Aroya, N., Meidan, R., Koch, Y., 1984. A differential effect of trypsin on pituitary gonadotropin-releasing hormone receptors from intact and ovariectomized rats. Evidence for the existence of two distinct receptor populations. Eur. J. Biochem. 140, 191–197. Moore-Ede, M., Sulzman, F., Fuller, C., 1982. Characteristics of circadian clocks. In: Moore-Ede, M., Sulzman, F., Fuller, C. (Eds.), The Clocks That Time Us. Harvard University Press, Cambridge.
125
Schirman-Hildesheim, T.D., Bar, T., Ben-Aroya, N., Koch, Y., 2005. Differential gonadotropin-releasing hormone (Gnrh) and Gnrh receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle. Endocrinology 146, 3401– 3408. Sellix, M.T., Egli, M., Henderson, R.P., Freeman, M.E., 2004. Ovarian steroid hormones modulate circadian rhythms of neuroendocrine dopaminergic neuronal activity. Brain Res. 1005, 164–181. Snabes, M.C., Kelch, R.P., Karsch, F.J., 1977. A daily neural signal for luteinizing hormone release in the untreated ovariectomized rat: changes in gonadotropin-releasing hormone content of the preoptic area and hypothalamus throughout the day. Endocrinology 100, 1521– 1525. Soares Jr., J.M., Masana, M.I., Ersahin, C., Dubocovich, M.L., 2003. Functional melatonin receptors in rat ovaries at various stages of the estrous cycle. J. Pharmacol. Exp. Ther. 306, 694–702. Van Der Beek, E.M., 1996. Circadian control of reproduction in the female rat. Prog. Brain Res. 111, 295–320. Van Der Beek, E.M., Horvath, T.L., Wiegant, V.M., Van Den Hurk, R., Buijs, R.M., 1997. Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: combined tracing and light and electron microscopic immunocytochemical studies. J. Comp. Neurol. 384, 569–579. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (7), 34.1–34.12. Williams, L.M., 1989. Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography. J. Mol. Endocrinol. 3, 71–75. Yoo, S.H., Yamazaki, S., Lowrey, P.L., Shimomura, K., Ko, C.H., Buhr, E.D., Siepka, S.M., Hong, H.K., Oh, W.J., Yoo, O.J., Menaker, M., Takahashi, J.S., 2004. Period2::luciferase real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 101, 5339–5346.
Daily GnRH and GnRH-receptor mRNA expression in the ovariectomized and intact rat Tamar D. Schirman-Hildesheim, Nurit Ben-Aroya, Yitzhak Koch ∗ Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel
Abstract We recently described patterns of GnRH and GnRH receptor (GnRH-R) expression in the hypothalamus, pituitary and ovary throughout the rat estrus cycle. Here, we wished to distinguish between regulatory effects of ovarian factors and underlying circadian rhythmicity. We quantified GnRH and GnRH-R mRNA in the pituitary and hypothalamus of long-term ovariectomized (OVX) rats, at different times of day, using real-time PCR. Furthermore, we expanded our previous study of hypothalamic and pituitary GnRH and GnRH-R expression in intact rats by including more time points throughout the estrus cycle. We found different daily patterns of GnRH and GnRH-R expression in intact versus OVX rats, in both tissues. In the hypothalamus of OVX rats, GnRH mRNA peaked at 12, 16 and 20 h, whereas in the hypothalamus of intact rats we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred at 17 h. In this tissue, GnRH-R fluctuated less significantly and peaked at 16 h in OVX rats. During the estrus cycle, we observed higher levels in the afternoon of each day except on estrus. In OVX rats, pituitary GnRH mRNA rose sharply at 9 h, with low levels thereafter. In these animals, pituitary GnRH-R also peaked at 9 h followed by a second rise at 22 h. In intact rats pituitary GnRH was high at noon of diestrus-II and on estrus, whereas GnRH-R mRNA was highest in the evening of diestrus-II. This is the first demonstration of daily GnRH and GnRH-R mRNA expression patterns in castrated animals. The observed daily fluctuations hint at underlying tissue-specific circadian rhythms. Ovarian factors probably modulate these rhythms, yielding the observed estrus cycle patterns. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: GnRH mRNA; GnRH receptor mRNA; Estrus cycle; Ovariectomized rats; Daily expression pattern; Rat hypothalamus; Rat pituitary
1. Introduction Reproductive functions in all vertebrates are orchestrated by the hypothalamic gonadotropin-releasing hormone, GnRH. This decapeptide is released from hypothalamic nerve endings into the portal system, leading it into the anterior pituitary, where it induces the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. GnRH and its receptor (GnRH-R) are also produced in a variety of extra-hypothalamic organs and glands, where their role has not been elucidated yet. We have recently described tissue-specific fluctuations in the levels of GnRH and GnRH-R mRNA in the hypothalamus, pituitary and ovary throughout the estrus cycle (Schirman-Hildesheim et al., 2005). Subsequently, we wished to discriminate between estrus cycle stage-dependant regulation by ovarian factors and underlying circadian oscillations in GnRH and GnRH-R expression.
∗
Corresponding author. Tel.: +972 8 9342790; fax: +972 8 9344131. E-mail address: [email protected] (Y. Koch).
0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.03.010
The circadian system plays a central role in mammalian reproduction (Goldman, 1999; Barbacka-Surowiak et al., 2003; Kennaway, 2005). In many spontaneously ovulating species, including rodents, ovulation and the preceding gonadotropin surge occur at roughly the same time in every cycle. Moreover, a shift in the timing of light onset leads to a corresponding shift in the timing of ovulation (Hoffmann, 1969). In a time-free environment (e.g. continuous dim light illumination) the estrus cycle maintains its circadian rhythmicity (Goldman, 1999). A daily neurogenic stimulus for luteinizing hormone (LH) surges has been demonstrated in rats by Everett and Sawyer (1949). The same researchers later showed that blocking of this neurogenic stimulus, using pentobarbital, delays the LH surge and ovulation by precisely 24 h (Everett and Sawyer, 1950). Later studies (Legan et al., 1975), using ovariectomized (OVX) rats, have pointed at the estrogen-dependency of these daily evening – centrally regulated – LH surges. In fact, steroid hormones have since been shown to play an important role in fine tuning the circadian clock (Abizaid et al., 2004, 2005; Sellix et al., 2004). Thus, mammalian reproduction is regulated by circadian mechanisms that are entrained to the light:dark cycles and are under the influence of ovarian factors.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
Central and peripheral circadian rhythms are classically thought to be regulated by the suprachaismatic nucleus (SCN) of the hypothalamus, which is itself entrained to the pattern of daylight. The SCN has been shown to be anatomically connected to GnRH-producing neurons in the hypothalamus (Van Der Beek et al., 1997). Recent evidence however, supports the contention that peripheral tissues and sites outside of the SCN, including GnRH-producing neurons (Gillespie et al., 2003), are capable of self-sustaining circadian rhythms even in the absence of central coordination (Yoo et al., 2004). Furthermore, since melatonin receptors are present in various tissues outside the brain, such as the ovary (Soares et al., 2003) and pituitary (Williams, 1989), synchronization of circadian rhythms with daylight could be achieved locally. In the present study, we searched for daily, ovarianindependent, variations in the levels of GnRH and GnRH-R mRNA, which might underlie the tissue-specific patterns of expression observed throughout the estrus cycle. To this end, we abolished the regulatory influence of ovarian factors by ovariectomizing adult rats. We then measured levels of pituitary and hypothalamic GnRH and GnRH-R expression during different times of day. Indeed, we found that GnRH and GnRH-R mRNA concentrations fluctuate during the day, in a tissue-specific and ovarian-independent manner. Moreover, we extended our previous study of estrus cycle patterns in GnRH and GnRH-R expression by studying additional time points during diestrus-I, -II and estrus, so as to present a comparison of daily expression patterns in intact versus OVX rats. 2. Materials and methods 2.1. Animals All animals were purchased from Harlan Laboratories (Rehovot, Israel) and all experiments were carried out in compliance with the regulations of the Weizmann Institute of Science and using accepted standards of humane animal care. Seven to nine weeks old female Wistar rats were used. Animals were housed under constant conditions of temperature and humidity with lights on between 6 a.m. and 8 p.m. and food available ad libitum. For the intact rat study, estrus cycles were monitored via vaginal smears and only rats showing at least three consecutive 4-day cycles were used. Animals were sacrificed by decapitation at the times listed in Table 1. For the OVX rat study, 7–8-week old Wistar rats were ovariectomized under anesthesia (Ketamine HCl and diazepam, 7.5 and 0.375 mg per 150 g animal, respectively). One month later the animals were sacrificed by decapitation at the times listed in Table 1. After decapitation, tissues were immediately removed and placed in 10 volumes of RNA Later (Ambion, Austin, TX, USA) until subsequent RNA extraction. The hypothalamus was dissected out to a depth of ∼3 mm with the following borders: the anterior edge of the optic chiasm, the anterior edge of the mammillary bodies and the two hypothalamic sulci on either lateral side. Both ovaries were removed and oviducts were examined for the presence of ova only in the groups sacrificed in the morning of estrus. OVX rats were examined to ascertain the absence of ovary fragments. The anterior pituitary was removed together with the attached posterior lobe.
121
Table 1 Times of animal sacrifice and sample sizes Time of day 06:30 07:30 09:00 10:00 12:00 14:00 15:00 16:00 17:00 19:00 20:00 21:30 22:00
Diestrus-I (n) Diestrus-II (n) Proestrus (n) Estrus (n) OVX (n)
4 12 5
10 6
4
4
6
7
10 4 4 5 8 10
7
6 6 8
5
5 5
5 5
7 5 5 5
6 6
ethanol and loaded onto an RNEasy mini-column (Qiagen GmbH, Hilden, Germany). The procedures for RNA isolation and purification, as well as on-column DNAse treatment (Qiagen), were then carried out as detailed in the manufacturer’s instructions. RNA samples were eluted in nuclease-free water (Qiagen). RNA concentration was quantified using a NanoDrop machine (NanoDrop Technologies, Wilmington, Delaware, USA) and its RNA purity was assessed on the same machine using the 260:280 and 260:230 nM ratios. All samples had 260:280 nM ratios between 1.8 and 2.1, and 260:230 nM ratios above 1.7. RNA integrity was assessed by examining the 28S and 18S bands of representative samples loaded onto a 1.5% agarose gel stained with ethidium bromide.
2.3. Reverse transcription (RT) For each tissue, equal amounts of all RNA samples were reversetranscribed simultaneously. Hypothalamic RNA samples (2 g each) were reverse-transcribed using M-MLV RNase H+ (Promega, Madison, WI, USA) according to the manufacturers’ instruction. Each reaction contained: 0.5 g Oligo-dT (Amersham Biosciences, Piscataway, NJ, USA), 0.52 mM of each dNTP (MBI Fermentas, St. Leon-Rot, Germany), 25 units RNAguard RNase inhibitor (Amersham Biosciences), 5 l of the 5× M-MLV reaction buffer (Promega) and 200 units of the enzyme in a total volume of 25 l. Pituitary RNA samples (4 g each) were reverse-transcribed using SuperScript II RNase H− Reverse Transcriptase kit (Invitrogen Life Technologies, Carlsbad, CA, USA). Each 20 l reaction contained: 0.5 g Oligo-dT (Amersham Biosciences), 0.5 mM of each dNTP (MBI Fermentas, Germany), 40 units porcine liver ribonuclease-inhibitor (Takara Bio Inc., Shiga, Japan), 2 l 0.1 M DTT, 4 l of the 5× first-strand buffer (Invitrogen) and 40 units of the enzyme. All RT reactions were performed at 42C and contained a negative control, which consisted of nuclease-free water instead of RNA. The linearity of the RT reaction was evaluated as described previously (Schirman-Hildesheim et al., 2005).
2.4. Gene-specific primers and taqman hybridization probes Primers were designed on two different exons so as to span one intronic sequence. TaqMan hybridization probes were designed to span an exon–exon junction (TIB-Molbiol, Berlin, Germany). All primer and probe sequences, PCR product sizes, product identity and annealing temperatures have been reported previously (Schirman-Hildesheim et al., 2005).
2.5. Relative real-time PCR 2.2. RNA purification Tissues were removed from the RNA later, weighed and homogenized in 0.5 mL Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). Following the addition of 100 l chloroform and phase separation by centrifugation (at 4C), the aqueous layer was washed with an equal volume of 70%
All real-time PCR reactions were carried out exactly as detailed elsewhere (Schirman-Hildesheim et al., 2005). Each real-time PCR reaction included a no-template control (NTC) as well as five or six serial four-fold dilutions, in duplicates, of a cDNA pool containing all experimental samples of the respective tissue. The pre-normalized DNA quantity of each gene in every sample was
122
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
estimated relative to this dilution series. This dilution series also served to assess the reaction performance (E and R2 ). The threshold cycle (Ct) was set so as to obtain the highest reaction efficiency and correlation coefficient. For normalization of gene expression, a panel of four candidate reference genes (HPRT, Cyclophillin, RPL19 and -actin) were tested in all experimental samples in order to identify the most stably expressed genes in all tested times in both intact and OVX rats. Primer sequences and reaction conditions for all tested reference genes, as well as the criteria used to select the most stable reference genes, have been described previously (ibidem). Since not all samples of a particular tissue or condition (intact versus OVX) could be assayed simultaneously for each gene, a common set of at least five ‘calibrator’ samples was included in every reaction for inter-reaction calibration (see below). Additionally, in order to compare between expression levels in OVX rats versus intact rats, each qPCR reaction with OVX samples also contained a set of samples from the same tissue of intact animals sacrificed at 10 h of diestrus-I. The latter samples served to calibrate the relative mRNA levels in OVX rats to those in intact rats (see below).
have been normalized to that at 10 h of diestrus-I, as explained in Section 2 (Section 2.6). It is important to note however, that in order to quantitatively compare the relative mRNA amounts in intact versus OVX rats, one must assume that the reference genes’ mRNA, used for normalization, are equally abundant in both conditions. 3.2. Relative daily levels of GnRH expression in intact and OVX rats
3. Results
Significant daily fluctuations in the relative expression levels of GnRH were found in intact as well as OVX rats in both the hypothalamus and pituitary (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.05). In the hypothalamus of intact rats (Fig. 1A, top graph), non-significant variations were observed during diestrus-I and estrus, though evening levels seem higher on both days, as compared to morning levels. During diestrusII and proestrus, significantly high levels were found in the evening, at 19 and 17 h, respectively. In the hypothalamus of OVX rats (Fig. 1A, bottom graph), low mRNA levels were detected at 14, 17, 19 and 22 h, whereas peak levels were recorded at 12, 16 and 20 h. Overall, similar relative amounts of GnRH mRNA were measured in intact and OVX rats, if one assumes equal abundance of RPL19 and Cyclophillin in both conditions. In the pituitary of intact rats (Fig. 1B, top graph), GnRH mRNA expression remained rather stable on diestrus-I. Significant fluctuations occurred during diestrus-II and proestrus. On the former, peak levels were measured at noon, with low levels in the afternoon. On proestrus, GnRH mRNA was at a lull in the morning and fluctuated thereafter. GnRH levels were highest throughout the estrus day as compared to all other times of the cycle (except for noon of diestrus-II), and remained stable during this day. In the pituitary of OVX rats (Fig. 1B, bottom graph) we noted a rather different daily GnRH expression pattern, with a stark rise at 9 h (eight-fold increase) followed by much lower levels (estrus cycle levels) throughout the remainder of the day.
3.1. Stably expressed reference genes in the pituitary and hypothalamus of intact and OVX rats
3.3. Relative daily levels of GnRH-R expression in intact and OVX rats
In our hands, Cyclophillin and RPL19 were the most stably expressed genes across all hypothalamic and pituitary samples of intact rats as well as in hypothalamic samples of OVX rats. Cyclophillin and -actin were found to be the most stably expressed reference genes among pituitary samples of OVX rats. Details regarding the selection criteria used have been described elsewhere (Schirman-Hildesheim et al., 2005). The two most stably expressed reference genes were used to normalize the expression of GnRH and GnRH-R in each sample, as described in Section 2. Mean relative expression values (±standard error of the mean, S.E.M.) in each time group in both experimental conditions (OVX versus intact) are plotted in Fig. 1. The daily expression patterns in OVX animals are quadruplicated in the graphs, so as to cover a range of 4 days, enabling an easier comparison to each day of the estrus cycle. All expression levels
Statistical analysis of hypothalamic GnRH-R expression pattern across the estrus cycle (Fig. 1C, top graph) reveals only non-significant variations, possibly due to relatively high interindividual variability (ANOVA, p > 0.05). Nonetheless, significant local variations can be noted: (i) higher levels at 16 h versus 12 h on diestrus-I, (ii) higher levels at 12 h versus 10 h on diestrus-II (Student’s t-test, p < 0.05). Moreover, on proestrus, GnRH-R mRNA peaked at 14 and 21 h. On the day of estrus, GnRH-R mRNA seemed to remain constant. In the hypothalamus of OVX rats (Fig. 1C, bottom graph), comparison of each pair of time points using Student’s t-test resulted in significant differences at 16 h as compared to 9 or 17 h. Overall, GnRH-R mRNA concentration was slightly higher in OVX rats as compared to intact rats, assuming equal abundance of the reference genes.
2.6. Data analysis and statistics The relative amounts of GnRH or GnRH-R mRNA were calculated in the following manner: (i) the pre-normalized DNA quantity in each sample (obtained from the dilution series) was divided by the geometric mean of the DNA quantities of the calibrator samples (see above) to account for inter-reaction differences; (ii) this calibrated DNA quantity in each sample was then divided by the geometric mean of the DNA quantities of the two most suitable reference genes (Vandesompele et al., 2002), to produce a ‘normalized DNA quantity’; (iii) the mean normalized DNA quantity was computed for each time group, in every tissue; (iv) finally, every mean normalized DNA quantity was normalized to that of the diestrus-I 10 h group (assayed in the same qPCR run, as described above) to obtain comparable results from intact and OVX samples. The final values obtained are hereafter referred to as the ‘relative expression levels’ of GnRH or GnRH-R. All statistical analyses were performed using JMP IN Statistical Discovery Software, Version 5.1 (SAS Institute Inc., Cary, NC, USA). For each tissue and gene the distribution of the obtained relative amounts of GnRH and GnRH-R mRNA in all samples were tested for normality using the Kolmogorov–Smirnov–Lilliefor’s test. Statistical evaluation of differences between time groups was performed using both parametric and non-parametric multiple comparison tests (one-way ANOVA, and the Wilcoxon rank-sum test, respectively), followed by pairwise comparisons of means using the least significant difference (LSD) test and the Student’s t-test at confidence level of 95%.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
123
Fig. 1. Daily expression patterns of GnRH and GnRH-R mRNA in the hypothalamus and pituitary of intact and ovariectomized rats. GnRH expression in the hypothalamus of intact (A, top panel) and OVX rats (A, bottom panel); GnRH expression in the pituitary of intact (B, top panel) and OVX rats (B, bottom panel); GnRH-R expression in the hypothalamus of intact (C, top panel) and OVX rats (C bottom panel); GnRH-R expression in the pituitary of intact (D, top panel) and OVX rats (D, bottom panel). Numbers on the abscissa represent hours of the day. The days of the estrus cycle is indicated above the top panel in each graph. The daily expression patterns detected in OVX animals are quadruplicated in the graphs, so as to cover a range of 4 days, enabling an easier comparison to each day of the estrus cycle. Values are mean mRNA amounts ± S.E.M. (arbitrary values) of 4–12 rats per time group, as indicated in Table 1. Relative mRNA expression levels were calculated as described in Section 2 (Sections 2.5–2.6). Different letters indicate statistically significant differences between groups (Student’s t-test, p < 0.05).
In the pituitary, we observed significant variations in the level of GnRH-R mRNA across the estrus cycle (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.0001; Fig. 1D, top graph) as well as on each day except for estrus, when they were low but stable. Thus, on diestrus-I, GnRH-R mRNA was most abundant at 10 h, lowest at 19 h, with a lower peak at 14 h. A rather similar pattern was observed in the pituitary of OVX rats (Fig. 1D,
bottom graph), where high levels occurred at 9 h, and low levels at 19 h, with an intermittent peak at 14 h (one-way ANOVA and Wilcoxon Rank-Sums test, p < 0.05). In OVX rats, GnRH-R was also high at 22 h. We did not sample this time in intact rats. On diestrus-II, GnRH-R was expressed at highest levels in the evening, at 19 h, which was also its estrus cycle peak time. On proestrus, pituitary GnRH-R expression was low in the morn-
124
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125
ing and fluctuated during the remainder of the day, much like its ligand. It appears that, generally, GnRH-R mRNA is more abundant in OVX versus intact rats (3–12-fold difference), assuming equal reference genes abundance. 4. Discussion To the best of our knowledge, this is the first demonstration of a GnRH and GnRH-R mRNA expression pattern in the pituitary and hypothalamus of castrated animals. Snabes et al. (1977) have previously demonstrated daily changes in hypothalamic GnRH content in untreated OVX rats, such that higher concentrations were observed in the morning versus the afternoon. The mRNA expression pattern we found is quite different and involves fluctuations throughout the day, with a peak almost every 4 h (Fig. 1A, bottom). Interestingly, we observed somewhat higher GnRH mRNA concentrations at 19 h on every day of the estrus cycle except proestrus, when the peak occurred slightly earlier, at 17 h (Fig. 1A, top). Similarly, in OVX animals GnRH expression was also found to be higher in the evening, at 20 h, slightly later than in intact animals. It is possible that ovarian factors, such as estrogen, serve to advance the circadian clock governing hypothalamic GnRH production. In support of this hypothesis, estrogen has recently been shown to modulate hypothalamic expression of transcription factors, which precede the expression of clock gene expression in the SCN (Abizaid et al., 2004). During the estrus cycle, we noted an interesting – though weak – hypothalamic GnRH-R expression pattern, consisting of higher levels in the afternoon of each day except on estrus (Fig. 1C, top). Thus, GnRH-R rose at 16 h on diestrus-I, paralleling a peak in OVX rats (Fig. 1C, bottom), and at 12 and 14 h of diestrus-II and proestrus, respectively. This pattern should further be confirmed and its physiological significance is to be unraveled. It is noteworthy however, that the rhythmic expression of hypothalamic GnRH and GnRH-R does not necessarily depend on innervations from the SCN since clock genes have been reported to be expressed within GnRH-producing neurons themselves, in vitro (Gillespie et al., 2003). The daily GnRH expression pattern we observed in the pituitary of OVX rats is rather striking, with a clear peak at 9 h (Fig. 1B, bottom). This pattern is not paralleled on any day of the estrus cycle (Fig. 1B, top). As we mentioned earlier, high hypothalamic GnRH concentrations in OVX rats have previously been recorded in the morning (Snabes et al., 1977). One could tentatively speculate that the higher morning hypothalamic GnRH content is accompanied by higher release into the portal system, which might positively feedback upon the pituitary GnRH-synthesizing cells. An alternative possibility stems from the recent demonstration that pituitary cells are capable of self-sustained circadian oscillations for several days in isolation (Yoo et al., 2004). Thus, GnRH expression in pituitary cells could be governed by local oscillatory molecular mechanisms with a circadian rhythm. The latter could be entrained to daylight via melatonin signaling (Williams, 1989). Pituitary GnRH-R mRNA levels in OVX rats also seem to be higher in the morning, at 9 h (Fig. 1D, bottom). Unlike GnRH,
high levels of GnRH-R expression were also recorded at 14 and 22 h. Though we did not sample intact animals at 22 h on each day of the estrus cycle, the expression pattern obtained on diestrus-I resembles that of OVX rats. On other days of the cycle, the daily expression pattern is quite different (Fig. 1D, top). The physiological significance of these observations is not clear. Interestingly however, GnRH-R mRNA concentrations seem, overall, higher in OVX than in intact rats, as previously demonstrated (Kakar et al., 1994). A similar phenomenon has also been demonstrated at the protein level (Liscovitch et al., 1984). We provide here a more elaborate outline of hypothalamic and pituitary GnRH and GnRH-R expression throughout the estrus cycle, to supplement our previous report (Schirman-Hildesheim et al., 2005). Of note are the following new observations, which we cannot at this time correlate with circadian features seen in OVX rats: (i) high concentrations of pituitary GnRH mRNA throughout estrus, relative to all other days in the cycle (except 14 h of diestrus-II; Fig. 1B, top); (ii) a sharp increase in pituitary GnRH-R production in the evening of diestrus-II, which probably serves to prepare, prime, the gonadotropes for the following day’s hormonal surge (Fig. 1D, top). These observations are to be further confirmed and interpreted in terms of biological significance. We have previously described tissue-specific estrus cycle expression patterns of GnRH and its receptor, and suggested that these genes are locally regulated in accordance with the animal’s reproductive state, or phase of the estrus cycle (Schirman-Hildesheim et al., 2005). Moreover, we speculated that GnRH might play a role in the preparation of the pituitary and ovary for the imminent preovulatory surge, possibly via local GnRH-gonadotropin axes. The contribution of underlying circadian fluctuations in GnRH and GnRH-R expression, which emerge in the absence of ovarian factors, to the estrus cycle patterns of these genes’ expression should be further investigated. It would be interesting to verify the dependence of the observed circadian expression patterns on the lighting periodicity and timing. Indeed, ‘true’ circadian rhythms display not only a circa-24 h periodicity, which is maintained in a time-free environment (e.g. constant dim light), but also entrainment to environmental light–dark cycles (MooreEde et al., 1982; Sellix et al., 2004). The evolutionary advantage of synchronizing reproductive functions to day time, such that ovulation and receptivity concur with the animal’s potential social interaction, is a well known phenomenon, which has been reviewed at length (Van Der Beek, 1996; Barbacka-Surowiak et al., 2003; Goldman, 1999). Acknowledgement This study was supported by the Nella & Leon Benoziyo Center for the Neurosciences and the German-Israeli Foundation for Scientific Research and Development (GIF). References Abizaid, A., Mezei, G., Horvath, T.L., 2004. Estradiol enhances light-induced expression of transcription factors in the Scn. Brain Res. 1010, 35–44.
T.D. Schirman-Hildesheim et al. / Molecular and Cellular Endocrinology 252 (2006) 120–125 Abizaid, A., Mezei, G., Thanarajasingam, G., Horvath, T.L., 2005. Estrogen enhances light-induced activation of dorsal raphe serotonergic neurons. Eur. J. Neurosci. 21, 1536–1546. Barbacka-Surowiak, G., Surowiak, J., Stoklosowa, S., 2003. The involvement of suprachiasmatic nuclei in the regulation of estrous cycles in rodents. Reprod. Biol. 3, 99–129. Everett, J.W., Sawyer, C.H., 1949. A neural timing factor in the mechanism by which progesterone advances ovulation in the cyclic rat. Endocrinology 45, 581–595, llust. Everett, J.W., Sawyer, C.H., 1950. A 24-hour periodicity in the “Lh-release apparatus” of female rats, disclosed by barbiturate sedation. Endocrinology 47, 198–218. Gillespie, J.M., Chan, B.P., Roy, D., Cai, F., Belsham, D.D., 2003. Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting Gt1-7 neurons. Endocrinology 144, 5285–5292. Goldman, B.D., 1999. The circadian timing system and reproduction in mammals. Steroids 64, 679–685. Hoffmann, J.C., 1969. Light and reproduction in the rat: effect of lighting schedule on ovulation blockade. Biol. Reprod. 1, 185–188. Kakar, S.S., Grantham, K., Musgrove, L.C., Devor, D., Sellers, J.C., Neill, J.D., 1994. Rat gonadotropin-releasing hormone (Gnrh) receptor: tissue expression and hormonal regulation of its Mrna. Mol. Cell Endocrinol. 101, 151–157. Kennaway, D.J., 2005. The role of circadian rhythmicity in reproduction. Hum. Reprod. Update 11, 91–101. Legan, S.J., Coon, G.A., Karsch, F.J., 1975. Role of estrogen as initiator of daily Lh surges in the ovariectomized rat. Endocrinology 96, 50–56. Liscovitch, M., Ben-Aroya, N., Meidan, R., Koch, Y., 1984. A differential effect of trypsin on pituitary gonadotropin-releasing hormone receptors from intact and ovariectomized rats. Evidence for the existence of two distinct receptor populations. Eur. J. Biochem. 140, 191–197. Moore-Ede, M., Sulzman, F., Fuller, C., 1982. Characteristics of circadian clocks. In: Moore-Ede, M., Sulzman, F., Fuller, C. (Eds.), The Clocks That Time Us. Harvard University Press, Cambridge.
125
Schirman-Hildesheim, T.D., Bar, T., Ben-Aroya, N., Koch, Y., 2005. Differential gonadotropin-releasing hormone (Gnrh) and Gnrh receptor messenger ribonucleic acid expression patterns in different tissues of the female rat across the estrous cycle. Endocrinology 146, 3401– 3408. Sellix, M.T., Egli, M., Henderson, R.P., Freeman, M.E., 2004. Ovarian steroid hormones modulate circadian rhythms of neuroendocrine dopaminergic neuronal activity. Brain Res. 1005, 164–181. Snabes, M.C., Kelch, R.P., Karsch, F.J., 1977. A daily neural signal for luteinizing hormone release in the untreated ovariectomized rat: changes in gonadotropin-releasing hormone content of the preoptic area and hypothalamus throughout the day. Endocrinology 100, 1521– 1525. Soares Jr., J.M., Masana, M.I., Ersahin, C., Dubocovich, M.L., 2003. Functional melatonin receptors in rat ovaries at various stages of the estrous cycle. J. Pharmacol. Exp. Ther. 306, 694–702. Van Der Beek, E.M., 1996. Circadian control of reproduction in the female rat. Prog. Brain Res. 111, 295–320. Van Der Beek, E.M., Horvath, T.L., Wiegant, V.M., Van Den Hurk, R., Buijs, R.M., 1997. Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: combined tracing and light and electron microscopic immunocytochemical studies. J. Comp. Neurol. 384, 569–579. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (7), 34.1–34.12. Williams, L.M., 1989. Melatonin-binding sites in the rat brain and pituitary mapped by in-vitro autoradiography. J. Mol. Endocrinol. 3, 71–75. Yoo, S.H., Yamazaki, S., Lowrey, P.L., Shimomura, K., Ko, C.H., Buhr, E.D., Siepka, S.M., Hong, H.K., Oh, W.J., Yoo, O.J., Menaker, M., Takahashi, J.S., 2004. Period2::luciferase real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 101, 5339–5346.