Molecular Human Reproduction, Vol. 9, No. 9, 503-507,
September 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Rhythmic expression of clock and clock-controlled genes in the rat oviduct
Submitted on March 5, 2003; accepted on May 2, 2003
Department of Obstetrics and Gynaecology, Reproductive Medicine Unit, University of Adelaide, Frome Road, Adelaide, South Australia, 5005, Australia
1 To whom correspondence should be addressed. e-mail: david.kennaway{at}adelaide.edu.au
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ABSTRACT |
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The rhythmic expression of clock and clock-controlled genes in the rat oviduct was investigated by real time RT–PCR. per1, per2, Clock, Bmal1, cry1 and cry2 were all expressed in the oviduct. With 4-hourly sampling over 24 h in a normal photoperiod, analysis of variance indicated that per2 and Bmal1 had highly significant sinusoidal-like changes with an amplitude of 3- and 10-fold respectively. Of the other clock genes, per1 and cry1 had non-significant rhythm amplitudes of 2.5- and 1.8-fold respectively. Using the same experimental approach the rhythmic expression of Bmal1, per1 and per2 mRNA in the liver was found to be highly significant with amplitudes of approximately 20-, 10- and 5-fold respectively. The expression of the clock-controlled transcription factors DBP and Rev-erb
showed significant rhythmicity in the oviduct with 5-fold changes in amplitude for both genes. Plasminogen activator inhibitor-1 (PAI-1), which has been implicated in oviduct function during the preimplantation period, also had a significant rhythm of expression (2.5-fold amplitude), peaking at the same time as the other clock-controlled genes, DBP and Rev-erb . These results show for the first time that the female reproductive tract is inherently rhythmic and suggests that the developing embryo may be subjected to rhythmic changes in the environment created by the oviduct during transition to the uterus. Key words: circadian/clock genes/E-box/embryo development/transcription factors
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Introduction |
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The suprachiasmatic nucleus (SCN) is generally recognized as being the master clock, controlling a wide range of circadian rhythms. Endogenous rhythmicity in the SCN is generated by negative and positive feedback control of a set of genes identified as ‘clock genes’. In recent years it has become clear that peripheral tissues also express the full range of these primary clock genes rhythmically. Subsequent microarray analyses have indicated that there are >500 slave or ‘clock-controlled’ genes rhythmically expressed in tissues such as the liver and heart (Storch et al., 2002). Comparisons between organs has shown that there is a relatively small proportion of these clock-controlled genes expressed rhythmically in all tissues (Delaunay and Laudet, 2002).
To date, rhythmicity in reproductive tissues has not been studied in any depth. The oviduct provides an optimal environment for embryo development through the secretion of ions, small molecules and proteins as well as controlling the timing of the passage of the embryo into the uterus prior to implantation. It thus plays an important role in embryo development, but is essentially made redundant in many assisted reproductive techniques. Instead embryos are cultured under conditions designed to mimic closely those in the oviduct using specialized media, growth factors, hormones, etc. No matter how good the media and culture conditions are, in-vitro culture cannot, however, mimic the changing conditions the embryo is exposed to in the oviduct. Consequently embryos developed in vitro develop slower than those in vivo (Harlow and Quinn, 1982) and there are epigenetic changes in the embryo which are thought to have serious long-term consequences (De Rycke et al., 2002; Thompson et al., 2002). A possibility not previously addressed is that the lack of rhythmicity in vitro during assisted reproduction may contribute to some of these problems. This is likely to be even more problematic with the trend towards longer culture times.
If the oviduct were to express daily rhythmicity in its secretion of growth factors, hormones or protease inhibitors, these changes may influence the rate of embryo development and the timing of gene expression during its transfer into the uterus. There is some preliminary evidence that the key clock genes per1, per2, cry1, cry2, Clock and Bmal1 are expressed in the mouse oviduct during early pregnancy but there are no reports of their temporal expression (Johnson et al., 2002). In this first study of rhythmicity in the oviduct, we have analysed the expression of the primary clock genes per1, per2, cry1, cry2, Clock and Bmal1 in the rat over a 24 h period by real time RT–PCR. In addition we have measured changes in expression, across the day, of clock-controlled transcription factors albumin D-element binding protein (DBP) (Ripperger et al., 2000) and Rev-erb (Preitner et al., 2002), the enzyme lactate dehydrogenase (LDH-A) (Rutter et al., 2001) and plasminogen activator inhibitor-1 (PAI-1) (Maemura et al., 2000).
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Materials and methods |
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Adult Wistar albino rats (150–170 g) were obtained from Laboratory Animal Services at the University of Adelaide where they had been maintained in a 12 h light:12 h dark photoperiod. On arrival in the Medical School Facility the rats were housed six to a cage under the same photoperiod. The animals were housed and treated according to the Australian Code of Practice for the care and use of animals for scientific purposes. The experiments were approved by the Animal Ethics Committee of the University of Adelaide. Four days after arrival, groups of six animals were killed by decapitation at 4-hourly intervals starting at 0700 (immediately after lights on).
Ovaries, oviduct and uteri as well as a section of liver were rapidly dissected and immediately placed in RNAlater® (Ambion, USA) at 4°C until processing. No formal attempt was made to obtain tissue at a specific stage of the cycle; however, all animals were cycling. At the time of dissection the ovaries were scored for the presence of fresh corpora lutea and colour and the uteri for evidence of edema. Statistical analysis by non-parametric analysis of variance (ANOVA) indicated that there were no significant differences between the time points.
Total oviduct and liver RNA was extracted using 200 µl TriReagent (Sigma Chemical Company, USA) according to the manufacturer’s protocol. Total RNA quantification was performed by taking an optical density reading at 260 nm. RNA (3 µg) in a volume of 20 µl was reverse-transcribed into first strand cDNA. The RNA was incubated with 2 µl of random hexamer primers (GeneWorks Pty Ltd, Australia) at 70°C for 10 min. The tubes were then cooled on ice for 5 min, and 8 µl of 5xRT buffer (Invitrogen, USA), 4 µl of 0.1 mol/l dithiothreitol (Invitrogen, USA) and 4 µl of 2'-deoxynucleoside 5'-triphosphates (Amersham Pharmacia Biotech, USA) added. The tubes were then incubated at 43°C for 2 min, and 2 µl of Superscript 1 Reverse Transcriptase (Invitrogen) was added. This was followed by incubation at 43°C for 90 min and 95°C for 5 min. 60 µl of ultrapure water was added to each sample to make a final volume of 100 µl of cDNA. The cDNA was then stored at –20°C until further use. Primers were designed with the ABI Prism Primer Express program (Applied Biosystems, USA) (see Table I for primers used). Amplification of cDNA was performed on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). To each well, a 2.5 µl aliquot of cDNA, 2 µl of 0.625 µmol/l forward and reverse primers, 3.5 µl of water and 10 µl of SYBR green (Applied Biosystems) were added. The samples were amplified by 1 cycle of 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, and 60°C for 1 min. Each sample was analysed in duplicate. Fluorescence was measured at the end of each cycle, and after 40 reaction cycles, a profile of fluorescence versus cycle number was obtained. An arbitrary threshold of fluorescence was set within the exponential phase of amplification (reference normalized = 0.1). The cycle at which the amplification of the product exceeded this threshold was determined and designated as cycle threshold (Ct).
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The expression of each gene within each sample was normalized against ß-actin, and expressed relative to a calibrator sample with the use of the formula 2–(
Ct) as described by K.Livak (PE-ABI, Sequence Detector User Bulletin 2). Ct can be defined as: [Ctgene of interest (unknown sample) – Ct ßactin (unknown sample)] – [Ctgene of interest (calibrator sample) – Ct ßactin (calibrator sample)]. The expression of ß-actin did not vary significantly across the six time points studied (ANOVA; P > 0.05). The calibrator sample was designated as the most highly expressed time point for each gene of interest, and therefore has an expression of 100%. The data were analysed by one-way ANOVA. |
Results |
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All six clock genes were expressed in oviduct tissue. They had cycle thresholds similar to the housekeeping gene and showed acceptable amplification efficiencies (Table II). They produced the expected exponential curves of fluorescence versus cycle number and the amplicons produced single bands of the appropriate sizes after gel electrophoresis. When the expression of each individual gene was subjected to ANOVA, per2 and Bmal1 showed highly significant sinusoidal-like changes over the 24 h sampling period with amplitudes of 3- and 10-fold respectively (Figure 1). Of the other clock genes, per1, cry1 and Clock also showed 2.5-, 1.8- and 2-fold changes in expression across the 24 h, but the rhythmicity was not statistically significant (Table II). Figure 2 shows the rhythmic expression of per1, per2 and Bmal1 in the liver. The timing of the peak in per1 mRNA expression at 1900 h and the nadir of Bmal1 expression at 1900 h are in close agreement with the timing of the expression of these genes in the oviduct. Similarly there is a rather broad peak in per2 expression in both tissues.
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The transcription factors DBP and Rev-erb
were also strongly expressed in the oviduct and both showed significant sinusoidal-like, 5-fold changes in expression across the 24 h period. Peak expression for both genes occurred around 1700 h. mRNA for the enzyme LDH-A and the protease inhibitor PAI-1 were strongly expressed in the oviduct. LDH-A expression did not change across the 24 h of sampling. In contrast, PAI-1 expression was clearly rhythmic (P < 0.05) with a peak occurring around 1700 h and a 2.5-fold rhythm amplitude.
All 10 genes were analysed in the same oviduct tissue samples; therefore, we investigated their interrelationships by multiple linear correlation analysis. Table III shows the correlation matrix and significance levels for each pair of genes. per1 expression was positively correlated with cry2, the two transcription factors DBP and Rev-erb and also with PAI-1 expression. per2 was positively correlated with cry2 and trended to a negative correlation with Bmal1 (P = 0.054). PAI-1 expression was positively correlated with per1, per2, cry2, DBP and Rev-erb .
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Discussion |
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This study has shown for the first time that clock genes in the oviduct are rhythmically expressed. Thus, per2 and Bmal1 were shown to peak
8 h apart in keeping with their primary roles in generating endogenous rhythms. The contemporary understanding of how rhythmicity is generated is that CLOCK/BMAL1 heterodimers initiate transcription of per1 and per2 (Shearman et al., 2000). PER1 and PER2 associate with CRY1 and CRY2 proteins to form heterodimer complexes along with the phosphorylating enzyme Casein Kinase 1E (Vielhaber et al., 2000) and return to the nucleus to inhibit the CLOCK/BMAL1 induction of per and cry genes. In the mouse it has been shown that PER2 also promotes Bmal1 transcription through mechanisms not yet fully understood, to provide a positive drive to the system (Yu et al., 2002). Rev-erb , itself a target for positive induction by CLOCK/BMAL1 inhibits Bmal1 induction (Preitner et al., 2002).
Thus the current study has shown that the oviduct not only has the appropriate elements but it also displays the appropriate temporal changes in clock gene expression implying functional rhythmicity. The demonstration of rhythmicity is an important finding since it has been recently reported that the testis expresses per1 and Bmal1 but at constant levels over 24 h (Morse et al., 2003). Rhythmic clock gene expression alone may mean very little if the cyclic transcription factors do not influence other target genes to alter function rhythmically. We therefore chose to investigate the expression of an additional four genes that have been shown in other organs to be expressed rhythmically by virtue of the canonical consensus circadian E-boxes in their promoters. Both DBP and Rev-erb are transcription factors that are rhythmic in liver (Ogawa and Ansai, 1995; Ueda et al., 2002) and SCN (Lopez-Molina et al., 1997; Ueda et al., 2002) with peak expression coinciding with per1 and in appropriate antiphase with Bmal1. In the rat oviduct, both were highly expressed and positively correlated with per1 and cry2.
The key metabolic enzyme lactate dehydrogenase is rhythmically expressed in brain (Rutter et al., 2001). In the rat oviduct, however, we failed to detect any suggestion of rhythmicity of LDH-A expression probably because its levels are determined by other factors in this tissue despite its circadian E-box. By contrast, the expression of the gene coding for the protease inhibitor PAI-1 was rhythmic, peaking between 1500 and 1900 h coincident with per1, cry2 and the transcription factors. PAI-1 has been shown to have a consensus circadian E-box and is rhythmically expressed in the heart. Moreover the rhythm of PAI-1 expression is greatly dampened in mice carrying the Clock 19 mutation, implying the critical role of the BMAL1/CLOCK heterodimer in its transcription. PAI-1 mRNA and protein have been detected in the oviduct previously in gilts (Kouba et al., 2000a;b) although we are not aware of any studies of its rhythmic expression. Kouba et al. proposed that PAI-1 may have a role in protecting the developing embryo from proteases and/or extracellular matrix remodelling of the oviduct and early embryo (Kouba et al., 2000b).
If other peripheral tissues such as the liver (Akhtar et al., 2002), heart (Storch et al., 2002) and cultured fibroblasts (Grundschober et al., 2001; Duffield et al., 2002) are a guide, it is likely that other genes besides PAI-1, for example cytokines and other growth factors, will be rhythmically expressed in the oviduct.
In this first study of clock gene rhythmicity in the oviduct, the tissue was not collected at a specific stage of the estrous cycle or pregnancy; instead cycling rats were chosen randomly at the various times of day. The numbers of animals killed at each time point prohibited any independent analysis of the effects of stage of cycle but is was clear that no stage was over-represented at a particular time of day. There is currently no evidence that clock genes have steroid receptor binding domains. In the brain, administration of estradiol to ovariectomized rats decreased the expression of cry2 but not cry1 at mid-light and mid-dark in the SCN, but no effects on rhythmicity were reported, nor were the effects of the steroid on the expression of any other clock genes studied (Nakamura et al., 2001). Finally our own studies on melatonin rhythms in rats across the estrous cycle indicated that any changes in the timing or amplitude of signals from the SCN to the pineal gland are very modest (White et al., 1997). Nevertheless future studies are needed to investigate whether the amplitude of the rhythm of clock and clock-controlled genes such as PAI-1 are influenced by the changing hormonal environment during the estrous cycle and the preimplantation period (Kouba et al., 2000b). Since it is also known that the function of the oviduct changes along its length, it would also be important to evaluate gene expression in the ampulla and isthmus regions.
The results reported here clearly cannot provide a definitive function of rhythmicity in the oviduct. Nevertheless we speculate that rhythmic secretion of proteins such as PAI-1 by oviductal epithelial cells may be one of the critical components in promoting timely embryo development in vivo. Despite advances in the understanding and refinement of culture conditions for in-vitro embryo culture, embryos still develop slower in vitro than in vivo. Moreover, in-vitro culture is known to increase the incidence of fetal abnormalities especially fetal growth disturbances. Further investigations of the role of rhythms during the pre-implantation period may provide the basis for improving embryo quality for IVF programmes.
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Acknowledgements |
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We thank Dr Hay Mook Kang, Cheongju University, Cheongju, Korea for providing the unpublished sequence for rcry1 and Dr Norio Ishida, National Institute of Bioscience and Human Technology, Higashi, Japan for providing the sequence for rper1 ahead of its GenBank submission. This study was supported in part by a grant from the Reproductive Medicine Unit.
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REFERENCES |
|---|
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TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Akhtar, R.A., Reddy, A.B., Maywood, E.S., Clayton, J.D., King, V.M., Smith, A.G., Gant, T.W., Hastings, M.H. and Kyriacou, C.P. (2002) Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus. Curr. Biol., 12, 540–550.[CrossRef][Web of Science][Medline]
De Rycke, M., Liebaers, I. and Van Steirteghem, A. (2002) Epigenetic risks related to assisted reproductive technologies: risk analysis and epigenetic inheritance. Hum. Reprod., 17, 2487–2494.
Delaunay, F. and Laudet, V. (2002) Circadian clock and microarrays: mammalian genome gets rhythm. Trends Genet., 18, 595–597.[CrossRef][Web of Science][Medline]
Duffield, G.E., Best, J.D., Meurers, B.H., Bittner, A., Loros, J.J. and Dunlap, J.C. (2002) Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells. Curr. Biol., 12, 551–557.[CrossRef][Web of Science][Medline]
Grundschober, C., Delaunay, F., Puhlhofer, A., Triqueneaux, G., Laudet, V., Bartfai, T. and Nef, P. (2001) Circadian regulation of diverse gene products revealed by mRNA expression profiling of synchronized fibroblasts. J. Biol. Chem., 276, 46751–46758.
Harlow, G.M. and Quinn, P. (1982) Development of preimplantation mouse embryos in vivo and in vitro. Aust. J. Biol. Sci., 35, 187–193.[Medline]
Johnson, M.H., Lim, A., Fernando, D. and Day, M.L. (2002) Circadian clockwork genes are expressed in the reproductive tract and conceptus of the early pregnant mouse. Reprod. Biomed. Online, 4, 140–145.[Medline]
Kouba, A.J., Alvarez, I.M. and Buhi, W.C. (2000a) Identification and localization of plasminogen activator inhibitor-1 within the porcine oviduct. Biol. Reprod., 62, 501–510.
Kouba, A.J., Burkhardt, B.R., Alvarez, I.M., Goodenow, M.M. and Buhi, W.C. (2000b) Oviductal plasminogen activator inhibitor-1 (PAI-1): mRNA, protein, and hormonal regulation during the estrous cycle and early pregnancy in the pig. Mol. Reprod. Dev., 56, 378–386.[CrossRef][Web of Science][Medline]
Lopez-Molina, L., Conquet, F., Dubois-Dauphin, M. and Schibler, U. (1997) The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior. EMBO J., 16, 6762–6771.[CrossRef][Web of Science][Medline]
Maemura, K., de la Monte, S.M., Chin, M.T., Layne, M.D., Hsieh, C.M., Yet, S.F., Perrella, M.A. and Lee, M.E. (2000) CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression. J. Biol. Chem., 275, 36847–36851.
Morse, D., Cermakian, N., Brancorsini, S., Parvinen, M. and Sassone-Corsi, P. (2003) No circadian rhythms in testis: Period1 expression is clock independent and developmentally regulated in the mouse. Mol. Endocrinol., 17, 141–151.
Nakamura, T.J., Shinohara, K., Funabashi, T. and Kimura, F. (2001) Effect of estrogen on the expression of Cry1 and Cry2 mRNAs in the suprachiasmatic nucleus of female rats. Neurosci. Res., 41, 251–255.[CrossRef][Web of Science][Medline]
Ogawa, H. and Ansai, Y. (1995) Diurnal rhythms of rat liver serine dehydratase, D-site binding protein, and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels are altered by destruction of the suprachiasmatic nucleus of the hypothalamus. Arch. Biochem. Biophys., 321, 115–122.[CrossRef][Web of Science][Medline]
Preitner, N., Damiola, F., Lopez-Molina, L., Zakany, J., Duboule, D., Albrecht, U. and Schibler, U. (2002) The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell, 110, 251–260.[CrossRef][Web of Science][Medline]
Ripperger, J.A., Shearman, L.P., Reppert, S.M. and Schibler, U. (2000) CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev., 14, 679–689.
Rutter, J., Reick, M., Wu, L.C. and McKnight, S.L. (2001) Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors. Science, 293, 510–514.
Shearman, L.P., Sriram, S., Weaver, D.R., Maywood, E.S., Chaves, I., Zheng, B., Kume, K., Lee, C.C., van der Horst, G.T., Hastings, M.H. et al. (2000) Interacting molecular loops in the mammalian circadian clock. Science, 288, 1013–1019.
Storch, K.F., Lipan, O., Leykin, I., Viswanathan, N., Davis, F.C., Wong, W.H. and Weitz, C.J. (2002) Extensive and divergent circadian gene expression in liver and heart. Nature, 417, 78–83.[CrossRef][Medline]
Thompson, J.G., Kind, K.L., Roberts, C.T., Robertson, S.A. and Robinson, J.S. (2002) Epigenetic risks related to assisted reproductive technologies. Short- and long-term consequences for the health of children conceived through assisted reproduction technology: more reason for caution? Hum. Reprod., 17, 2783–2786.
Ueda, H.R., Chen, W., Adachi, A., Wakamatsu, H., Hayashi, S., Takasugi, T., Nagano, M., Nakahama, K., Suzuki, Y., Sugano, S. et al. (2002) A transcription factor response element for gene expression during circadian night. Nature, 418, 534–539.[CrossRef][Medline]
Vielhaber, E., Eide, E., Rivers, A., Gao, Z.H. and Virshup, D.M. (2000) Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon. Mol. Cell. Biol., 20, 4888–4899.
White, R.M., Kennaway, D.J. and Seamark, R.F. (1997) Estrogenic effects on urinary 6-sulphatoxymelatonin excretion in the female rat. J. Pineal Res., 22, 124–129.[Web of Science][Medline]
Yu, W., Nomura, M. and Ikeda, M. (2002) Interactivating feedback loops within the mammalian clock: BMAL1 is negatively autoregulated and upregulated by CRY1, CRY2, and PER2. Biochem. Biophys. Res. Commun., 290, 933–941.[CrossRef][Web of Science][Medline]
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