This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cikos, S. Articles by Koppel, J. Search for Related Content PubMed PubMed Citation Articles by Cikos, S. Articles by Koppel, J.

Expression of adrenergic receptors in mouse preimplantation embryos and ovulated oocytes

tefan iko, Pavol Rehák, Soa Czikková, Jarmila Veselá and Juraj Koppel

Institute of Animal Physiology, Slovak Academy of Sciences, oltésovej 4, 04001 Koice, Slovakia

Correspondence should be addressed to iko; Email: cikos{at}saske.sk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Epinephrine and norepinephrine can play an important role in basic developmental processes such as embryogenesis and morphogenesis, regulating cell proliferation, differentiation and migration. We showed that ß-adrenergic receptors can mediate the effects of catecholamines on preimplantation embryos in our previous work. In the present study, we designed specific oligonucleotide primers which can distinguish among all members of the -adrenergic receptor family, and showed (using RT-PCR) that the 2C-adrenergic receptor is transcribed in ovulated oocytes, 8- to 16-cell morulae and expanded blastocysts. We did not detect the 2C-adrenoceptor transcript in 4-cell embryos. Our immunohistochemical study showed the presence of -2C-adrenoceptor protein in ovulated oocytes, 8- to 16- cell embryos and blastocysts, but the signal in 4-cell embryos was weak, and probably represents remaining protein of maternal origin. We did not detect any other -adrenergic receptor in preimplantation embryos and oocytes. Exposure of mouse preimplantation embryos to the 2-adrenergic agonist UK 14 304 led to significant reduction of the embryo cell number, and the effect was dose dependent. Our results suggest that epinephrine and norepinephrine could affect the embryo development in the oviduct via adrenergic receptors directly and support the opinion that maternal stress can influence the embryo even in very early pregnancy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Epinephrine and norepinephrine are well-known neurotransmitters and hormones that play an essential role in the regulation of the cardiovascular system and energy metabolism, especially in the case of stress or increased energy need. In addition to these well-known effects, epinephrine and norepinephrine can also play an important role in basic developmental processes such as embryogenesis and morphogenesis, regulating cell proliferation, differentiation, and migration (for review, see Buznikov et al. 1996, Pendleton et al. 1998, Weiss et al. 1998, Herlenius & Lagercrantz 2001). The essential role of epinephrine and norepinephrine in mouse fetal development has been clearly demonstrated, when mice lacking the capability to synthesize these compounds died in utero or shortly after birth (Kobayashi et al. 1995, Thomas et al. 1995, Zhou et al. 1995).

In our previous work, we demonstrated the possibility of direct influence of epinephrine and norepinephrine on the very early embryo (before its implantation into the uterus) via three subtypes of ß-adrenergic receptors (iko et al. 2005). The ß-adrenoceptors belong, together with 1- and 2- adrenoceptors, to the adrenergic receptor family of G protein-coupled receptors. To transduce extracellular signals, the receptors couple to heterotrimeric GTP-binding (G) proteins which activate various effectors. The ß-adrenoceptors couple predominantly to Gs proteins (G proteins which stimulate adenylyl cyclase activity), 1-adrenoceptors couple predominantly to Gq proteins (G proteins which stimulate phospholipase C activity) and 2-adrenoceptors couple predominantly to Gi proteins (G proteins which inhibit adenylyl cyclase activity). Signaling pathways leading from adrenergic receptors then regulate the activity of metabolic enzymes, ion channels or transcription factors (for review see, Watson & Arkinstall 1994, Saunders & Limbird 1999). Moreover, the adrenergic receptors can activate mitogen-activated protein kinases and thereby influence fundamental cellular processes (Alblas et al. 1993, DeGraff et al. 1999, Schramm & Limbird 1999, Kim et al. 2002, Shizukuda & Buttrick 2002, Pullar & Isseroff 2003).

To ascertain whether epinephrine and norepinephrine could influence the preimplantation embryo also via -adrenergic receptors, in the present study we examined the expression of all subtypes of 1- and 2- adrenergic receptors in mouse embryos at various stages of preimplantation development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Informatics analysis and primer design
Nucleotide sequences of mouse adrenergic receptors were obtained from public databases (GenBank, available at http://www.ncbi.nlm.nih.gov/ and GPCRDB: Information system for G protein-coupled receptors, available at http://www.gpcr.org/7tm/). MRNA nucleotide sequences of 1A-, 1B-, 1D-, 2A-, 2B-, and 2C- adrenergic receptors (GenBank accesion numbers: AF031431 [GenBank] , XM _126326, S80044 [GenBank] , M99377 [GenBank] , M94583 [GenBank] , M97516 [GenBank] ) were aligned using Clustal W (Higgins et al. 1996) and Blast 2 algorithms (Altschul et al. 1997). The regions which do not exert significant nucleotide homology among the members of the -adrenergic receptor family were selected for location of primers. The primers were designed using the program Primer 3 (available at http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and analysis of appropriate PCR products for recognition sites of restriction enzymes was performed using the program Webcutter 2.0 (available at http://rna.lundberg.gu.se/cutter2/).

Embryo recovery
All animal experiments were approved by the Ethical Committee of the Institute of Animal Physiology SAS, Koice. Female mice (ICR strain, Velaz, Prague, Czech Republic; 4–5 weeks old) underwent superovulation treatment by i.p. injection of 5 IU of serum gonadotropin (Folligon, Intervet International Bv. Boxmeer, Holland), followed 46 h later by administration of 5 IU of human chorionic gonadotropin (hCG, Organon, Oss, Holland). The mice were killed by cervical dislocation 24 h after hCG and unfertilized oocytes (at metaphase II) were isolated by flushing from the oviduct. To obtain preimplantation embryos, females were mated with males of the same strain overnight (mating was confirmed by identification of a vaginal plug), killed by cervical dislocation (57-, 72-, or 98 h after hCG) and the embryos (4-cell embryos, 8–16-cell morulae and expanded blastocysts) were isolated by flushing from the oviduct or uterus. Oocytes and embryos were washed in several drops of flushing-holding (FHM) medium (Lawits & Biggers 1993) containing 1% BSA and pooled according to their morphology. Cumulus cells were removed with 0.1% hyaluronidase (Sevac, Prague, Czech Republic).

RT-PCR
Total RNA was extracted from batches of 100–150 mouse preimplantation embryos and unfertilized oocytes. The RNA was also isolated from mouse tissues known to be rich in -adrenergic receptors (brain, heart and liver), which served as positive controls. TRIzol Reagent (Invitrogen Life technologies) was used according to the manufacturer’s instructions. Contaminating DNA in RNA preparations was digested by incubation for 15 min at 22 °C with amplification grade DNase I (Invitrogen Life technologies) in 20 mM Tris–HCl pH 8.4, 2 mM MgCl₂ and 50 mM KCl. The DNase I reaction was stopped by ethanol precipitation. RNA pellets were dissolved in water and denatured by incubation for 15 min at 65 °C.

The RNA was reverse transcribed at 42 °C for 1 h in 20 µl containing 200 units of Superscript II RNase H^– Reverse Transcriptase (Invitrogen Life technologies), 4 µM oligo dT18, 50 mM Tris–HCl pH 8.3, 3 mM MgCl₂, 75 mM KCl, 10 mM DTT, 500 µM dNTPs (dATP, dTTP, dCTP, dGTP), 40 units RNase OUT (Recombinant RNase Inhibitor, Invitrogen Life technologies) and 0.5 µg acetylated BSA. The reaction was terminated by heating at 95 °C for 5 min. To check for the presence of genomic DNA contamination in the RNA preparations, reverse transcriptase negative controls (no reverse transcriptase in the reaction) were carried out in parallel, using half from each RNA sample. The cDNA preparations were then cleaned by ethanol precipitation (Liss 2002) and the cDNA pellets were diluted in an appropriate amount of 10 mM Tris (pH 8.3) so that 1 µl of the cDNA corresponded to 2.5 embryo/oocyte equivalents.

PCR amplification was carried out in the presence of 0.5 µM of each oligonucleotide primer (see Table 1), 50 mM KCl, 10 mM Tris–HCl pH 8.3, 2 mM MgCl₂, 0.2 mM dNTPs (dATP, dTTP, dCTP, dGTP), and 0.05 units/µl Taq DNA polymerase (Invitrogen Life Technologies). One microliter of cDNA was amplified in 25 µl PCR mix. For 1B-, 1D-, 2A-, and 2B- adrenoceptors, an initial denaturation step at 95 °C for 2 min was followed by 40 cycles at 94 °C for 30 s and 68 °C for 90 s. For 1A-, and 2C- adrenoceptors an initial denaturation step at 95 °C for 2 min was followed by 40 cycles at 94 °C for 30 s, 63 °C for 45 s, and 72 °C for 45 s. To check for the presence of cross contamination, the reaction with water instead of cDNA was performed concurrently (blank reaction). Detection of ß-actin transcript using ß-actin primers (5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3', give a 539 bp PCR product; Temeles et al. 1994) served as a control for RNA integrity and the RT-PCR process.

Immunostaining
Preimplantation embryos and oocytes were isolated as described above and the zona pellucida was removed with 0.5% pronase in KSOM at 37 °C. Zona-free oocytes and embryos were washed thrice in PBS/BSA (PBS containing BSA; Sigma–Aldrich), fixed in methanol at 10 °C (Sigma–Aldrich) for 5 min, then washed in PBS/BSA, followed by washing in PBS/BSA/SAP (PBS/BSA containing 0.05% saponin (SAP); Sigma–Aldrich). Non-specific immunoreactions were blocked with 2.25% normal donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 0.1% saponin in PBS for 45 min at room temperature. The oocytes and embryos were incubated with the primary antibody raised against the 2C-adrenergic receptor (affinity-purified goat polyclonal antibody, 2C-AR C-20, Santa Cruz Biotechnology, 2 µg/ml) in PBS/BSA/SAP at 4 °C overnight. After extensive washing in PBS/BSA/SAP, specific secondary antibody coupled with fluorescein (donkey anti-goat IgG-FITC, Santa Cruz Biotechnology, 2 µg/ml) was used to visualize primary antibody reactions (30 min at room temperature). Afterwards, oocytes and embryos were washed in PBS/BSA/SAP and PBS/BSA, mounted on glass slides in UltraCruz mounting medium (Santa Cruz Biotechnology, contains DAPI for cell nuclei staining), sealed with coverslips and observed using an epifluorescent microscope (BX 51 Olympus, Tokyo, Japan).

Negative control groups of oocytes and embryos were incubated without the primary antibody or without the primary and secondary antibody, or with the primary antibody preadsorbed with an excess (20 times by weight) of the immunizing peptide according to the manufacturer’s protocol (Blocking peptide, 2C-AR C-20 P, Santa Cruz Biotechnology). Oocytes and embryos in each experimental group (incubation with primary antibody) were evaluated by comparison with control groups of oocytes and embryos.

Embryo culture and morphological evaluation
The four-cell embryos were isolated by flushing from the oviduct in FHM medium 57 h after hCG. They were washed thrice in KSOM culture medium, transferred into 30 µl drops of KSOM media supplemented with UK 14 304 (Sigma–Aldrich) dissolved in DMSO or with the equivalent volume of DMSO alone and cultured in a humidified atmosphere with 5.0% CO₂ at 37 °C for 60 h. Three doses of UK 14 304 were used: UK 14 304 was dissolved in DMSO and added to a KSOM culture medium (Lawits & Biggers 1993) at final concentrations of 0.1, 1, and 10 µM at the beginning of the incubation period and then 20 and 40 h later. The control groups of embryos were cultured in the presence of the equivalent amounts of solvent (DMSO) added into the KSOM at the same time as UK 14 304 (the compounds were added in the volume of 1 µl).

After the 60 h of incubation, the embryos were stained with Hoechst 33 342 (20 µg/ml; Sigma–Aldrich) for 10 min at 37 °C, washed, sealed with coverslips, and observed using an epifluorescent microscope (BX 51 Olympus). The number of cell nuclei was determined in all embryos. Each embryo was assigned to one of the four classes according to the cell number (developmental stage). Following classes were established: embryos with cell number less than 16, embryos with cell number between 17 and 32, embryos with cell number between 33 and 64, and embryos with cell number 65 and more. The distribution of embryos among the four developmental classes was then analyzed.

To eliminate experimental bias, at least three independent series were performed in each experimental group (the three UK 14 304 doses and the control group) and the results were pooled. The following total numbers of embryos (n) were examined: 10 µM UK 14 304 n = 139; 1 µM UK 14 304 n = 113; 0.1 µM UK 14 304 n = 140; DMSO controls n = 148.

The One-way ANOVA followed by Duncan’s test was used for evaluating the embryo cell number, while the chi-squared test was used for analyzing embryo distribution. Differences of P 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Primer design
We located the primers for -adrenergic receptors mainly in regions encoding a portion of the receptor outside of transmembrane domains, which are less conserved than transmembrane domains. The primers for 1D-adrenoceptor also detect an alternative splice variant coding longer adrenoceptor isoform (GenBank accession number XM_141369). The primers for 1A-adrenoceptor do not detect two potential alternative transcripts lacking a part of the coding sequence from the second exon (GenBank unfinished high-throughput cDNA sequences AK085653 [GenBank] and AK042759 [GenBank] , lastly modified in October 2006). Sequences and locations of the primers are shown in Table 1.

Amplification reaction for each primer pair was optimalized using positive controls – mouse tissues known to be rich in the receptor (Fig. 1a). The identity of each PCR product was verified by digestion with appropriate restriction enzymes according to the sequence information: digestion of the 1A-adrenoceptor PCR product (185 bp) with Alu I gave 112 and 73 bp DNA fragments, digestion of the 1B-adrenoceptor PCR product (140 bp) with Alu I gave 102 and 34 bp DNA fragments (the remaining 4 bp DNA fragment is not detectable on 2% agarose gel), digestion of the 1D-adrenoceptor PCR product (127 bp) with Hpa II gave 87 and 40 bp DNA fragments, digestion of the 2A-adrenoceptor PCR product (112 bp) with Hpa II gave 73 and 39 bp DNA fragments, digestion of the 2B-adrenoceptor PCR product (112 bp) with Taq I gave 74 and 38 bp DNA fragments, digestion of the 2C-adrenoceptor PCR product (105 bp) with Alu I gave 81 and 24 bp DNA fragments, and digestion with Mbo II gave 67 and 38 bp DNA fragments (Fig. 2).

View larger version (31K): [in this window] [in a new window]   Figure 1 RT-PCR with primers for -adrenergic receptors. (a) RT-PCR with primers for -adrenergic receptors in mouse positive control tissues. Agarose gels with separated PCR products are shown. Primers detecting 1A- (lane B, the 185 bp PCR product) and 1B- (lane C, the 140 bp PCR product) adrenoceptors were used with heart cDNA, primers detecting 1D- (lane D, the 127 bp PCR product), 2A- (lane E, the 112 bp PCR product), and 2C- (lane G, the 105 bp PCR product) adrenoceptors were used with brain cDNA, primers detecting 2B-adrenoceptor (lane F, the 112 bp PCR product) were used with liver cDNA. Lanes A and H, molecular weight markers (MWM). The sizes of the MWM in base pairs (bp) are indicated to the left and the right of the panel respectively. (b) RT-PCR analysis of 2C-adrenoceptor mRNA expression in mouse oocytes and preimplantation embryos. Agarose gels with separated PCR products (the 105 bp PCR product corresponds to the 2C-adrenoceptor) are shown. Lanes: B, oocytes; D, four-cell embryos; F, 8- to 16- cell embryos; H, blastocysts; J, positive control tissue (brain); C, E, G and I, corresponding reverse transcriptase negative controls; K, blank reaction. Lanes A and L, molecular weight markers (MWM). The sizes of the MWM in base pairs (bp) are indicated to the left and the right of the panel respectively.

View larger version (44K): [in this window] [in a new window]   Figure 2 Digestion of the PCR products with restriction enzymes. Lanes: B, digestion of the 1A-adrenoceptor PCR product with Alu I (112 + 73 bp DNA fragments); C, digestion of the 1B-adrenoceptor PCR product with Alu I (102 + 34 bp DNA fragments); D, digestion of the 1D-adrenoceptor PCR product with Hpa II (87 + 40 bp DNA fragments); F, digestion of the 2C-adrenoceptor PCR product with Alu I (81 + 24 bp DNA fragments); G, digestion of the 2C-adrenoceptor PCR product with Mbo II (67 + 38 bp DNA fragments); H, digestion of the 2A-adrenoceptor PCR product with Hpa II (73 + 39 bp DNA fragments); I, digestion of the 2B-adrenoceptor PCR product with Taq I (74 + 38 bp DNA fragments). Lanes A, E, and J, molecular weight markers (MWM). The sizes of the MWM in base pairs (bp) are indicated to the left and the right of the panel respectively.

Expression of -adrenergic receptor mRNAs

We detected a PCR product corresponding to the 2C-adrenergic receptor (105 bp) in oocytes, 8- to 16-cell morulae, and expanded blastocysts. No PCR product was detected in four-cell embryos or in the reactions where reverse transcriptase or cDNA were omitted (Fig. 1b). Digestion of the 105 bp PCR product with appropriate restriction enzymes produced DNA fragments of the expected sizes: digestion with Alu I gave 81 and 24 bp DNA fragments and digestion with Mbo II gave 67 and 38 bp DNA fragments (Fig. 2).

PCR products corresponding to other -adrenergic receptor mRNAs were not detected in the oocytes and embryos, even after an increase of cDNA amount in PCR or after an additional 25 cycles of reamplification (data not shown).

The embryos produced a PCR fragment corresponding to the ß-actin mRNA at all developmental stages, thus confirming the integrity of the RNA and the RT-PCR process (data not shown).

Immunohistochemical study
The immunoreactive 2C-adrenergic receptor was identified in oocytes, 8- to 16-cell morulae, and blastocysts, but the signal was weak in four-cell embryos (Fig. 3). The specificity of the signal was confirmed using several controls: the immunostaining intensity was significantly reduced in controls incubated with the primary antibody preadsorbed with the immunizing peptide (Fig. 3) or in controls incubated without the primary antibody or without the primary and secondary antibody (data not shown).

View larger version (90K): [in this window] [in a new window]   Figure 3 Immmunohistochemical localization of the 2C-adrenoceptor in mouse oocytes (A, B and C), four-cell embryos (D, E and F), 8- to 16-cell embryos (G, H and I), and blastocysts (J, K and L). Representative images are shown. A, D, G and J: Cell nuclei stained with DAPI; B, E, H and K: Oocytes/embryos incubated with the antibody against the 2C-adrenoceptor (identical samples as in images A, D, G and J); C, F, I and L: Oocytes/embryos incubated with the antibody against the 2-adrenoceptor preadsorbed with the immunizing peptide. The bar, 10 µm.

View larger version (8K): [in this window] [in a new window]   Figure 4 Embryo cell number after incubation with UK 14 304. The four-cell mouse embryos were incubated in the presence of indicated concentrations of UK 14 304 (UK) for 60 h and then the cell number was estimated. Values are arithmetical means + S.E.M. Numbers of embryos (n): 10 µM UK 14 304 n = 139; 1.0 µM UK 14 304 n = 113; 0.1 µM UK 14 304 n = 140; Control n = 148. aStatistical difference between the control group of embryos and the UK 14 304-treated groups: ***P < 0.001. bStatistical difference between the group of embryos treated with 10 µM UK 14 304 and the embryos treated with the two lower doses of UK 14 304: *P < 0.05, **P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

iko et al. 2005

Taken together, the results of our previous work (iko et al. 2005) and the results of the present study indicate that two types of adrenergic receptors are expressed in mouse ovulated oocytes and preimplantation embryos. The receptors are under the control of the same natural ligands (epinephrine, norepinephrine) but they couple primarily to G proteins with opposing actions on adenylyl cyclase activity (ß-adrenoceptors to Gs, 2-adrenoceptors to Gi). Co-stimulation of the two receptors in the same cell might be expected to antagonize each other’s signaling. However, the ability of a Gi protein-coupled receptor to decrease cAMP production in the cell is not automatic; it depends, for example, on the isoform of adenylyl cyclase present in the cell (for review, see Cooper 2003). Moreover, it has been shown that under certain circumstances 2-adrenoceptors can even stimulate cAMP production in some cell types, and the effect of 2-adrenoceptors on cAMP production appears to be dependent on agonist concentration (Duzic & Lanier 1992, Pepperl & Regan 1993, Eason & Liggett 1995, Pohjanoksa et al. 1997). An interesting cross-talk between 2-adrenoceptors and ß2-adrenergic receptor has been shown in pregnant rat myometrium with different receptor interactions at mid-pregnancy and in late pregnancy. By themselves, 2-adrenoceptor agonists did not significantly influence adenylyl cyclase activity in myometrial cells, but they were able to modulate the enzyme activity after ß2-adrenoceptor stimulation (Mhaouty et al. 1995, Limon-Boulez et al. 2001). The possibility of cross-talk between 2- and ß-adrenoceptors has also been shown in brain cells (Atkinson & Minneman 1992, Shivachar & Eikenburg 1999). These results indicate that simultaneous activation of ß- and 2- adrenergic receptors in the same cell need not necessarily have a contradictory effect on cAMP production; the signaling pathway employed by the receptor depends on the cell type and physiological status as well as on the intensity of receptor stimulation by a particular agonist.

To examine the effect of 2C-adrenoceptor activation on the development of mouse preimplantation embryos, we exposed the embryos to three doses of the 2-adrenergic agonist UK 14 304. We found that the percentage of embryos which reached higher developmental stages was significantly reduced after the exposure of the embryos to UK 14 304. Estimation of the mean embryo cell number revealed that the effect of UK 14 304 was dose dependent and led to significantly lower numbers of cells in UK 14 304-treated embryos in comparison with control embryos. These results indicate that activation of 2C-adrenoceptor in mouse preimplantation embryos can inhibit cell proliferation, similarly as shown for ß-adrenergic receptors (iko et al. 2005). The influence of adrenergic receptors on fundamental cellular processes has been demonstrated in various mammalian cell types, particularly regulation of cell proliferation, migration, and apoptosis by activated ß- and 2- adrenoceptors (Kennedy et al. 1983, Seuwen et al. 1990, Wang & Limbird 1997, Slotkin et al. 2000, Cussac et al. 2002, Kim et al. 2002, Shizukuda & Buttrick 2002, Pullar & Isseroff 2003, Zhu et al. 2003). Cell responses to stimulation of adrenergic receptors are mediated via various signaling pathways. The ß-adrenoceptors coupled predominantly to Gs proteins regulate the activity of metabolic enzymes, ion channels, or transcription factors. The 2-adrenoceptors coupled primarily to Gi proteins inhibit the activity of adenylyl cyclase and voltage-gated Ca²⁺ channels and activate receptor-operated K⁺ channels (for review see, Watson & Arkinstall 1994, Saunders & Limbird 1999). Moreover, stimulation or inhibition of mitogen-activated protein kinases by activated adrenergic receptors has been shown in several cell types utilizing a variety of signaling pathways (Alblas et al. 1993, DeGraff et al. 1999, Schramm & Limbird 1999, Lindquist et al. 2000, Hutchinson et al. 2002, Shizukuda & Buttrick 2002, Pullar & Isseroff 2003).

Natural ligands for adrenergic receptors, epinephrine and norepinephrine, have been detected in the oviductal fluid of some species. Concentrations of these catecholamines varied with the region of the oviduct and with the stage of the estrous cycle (Khatchadourian et al. 1987, Way et al. 2001). The possible sources of norepinephrine and epinephrine in the oviductal fluid could be nerves, blood circulation or graafian follicles (Helm et al. 1982, Fernandez-Pardal et al. 1986, Kannisto et al. 1986, Itoh et al. 2000, Kotwica et al. 2003). Moreover, it was speculated that catecholamines could be produced also by the early embryo itself (Burden & Lawrence 1973, Sadykova et al. 1990). Epinephrine and norepinephrine present in oviductal fluid could influence the oviduct epithelium via adrenergic receptors which have been shown in oviduct epithelial cells of several species (Tolszczuk & Pelletier 1988, Dickens et al. 1993, Einspanier et al. 1999). Increased fluid formation, effects on Cl^– ion transport and electrical potential differences have been shown after treatment of rabbit oviductal cells with agonists of adrenergic receptors (Dickens et al. 1993, Dickens & Leese 1994). Besides, the effects on the oviduct epithelium, adrenoceptor agonists can also influence the functioning of spermatozoa in the oviduct, including capacitation and acrosome reaction (Way & Killian 2002, Adeoya-Osiguwa et al. 2006).

It is well known that circulating levels of epinephrine and norepinephrine are highly elevated during stress and trauma. Animal experiments have convincingly demonstrated that prenatal maternal stress can significantly affect the pregnancy outcome (for review see Mulder et al. 2002). Some results suggest that maternal stress can negatively impact on the embryo even at very early stages of its development. For instance, it has been shown that litter sizes of female hamsters stressed during early pregnancy were significantly smaller than those of controls, with fetal loss occurring between days 5 and 10 of pregnancy (Pratt & Lisk 1989). In humans, it has been demonstrated that pregnancies characterized by increased maternal cortizol (commonly used stress marker) during the first three weeks after conception are more likely to result in spontaneous abortion. (Nepomnaschy et al. 2006).

Our results indicate that, besides ß-adrenergic receptors, the 2C-adrenoceptor is also expressed in mouse oocytes and preimplantation embryos. We also found that ligands for the receptors are capable of affecting the preimplantation embryo development in vitro. These results suggest that epinephrine and norepinephrine could directly affect embryo development in the oviduct via adrenergic receptors and support the opinion that maternal stress can influence the embryo even in very early pregnancy.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
This work was supported by Grant 2/6176/6 from the Slovak Academy of Sciences. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 4 January 2007
First decision 6 February 2007
Accepted 22 February 2007

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

Adeoya-Osiguwa SA, Gibbons R & Fraser LR 2006 Identification of functional 2- and ß-adrenergic receptors in mammalian spermatozoa. Human Reproduction 21 1555–1563.[Abstract/Free Full Text]

Alblas J, Van Corven EJ, Hordijk PL, Milligan G & Moolenaar WH 1993 G_i – mediated activation of the p21ras-mitogen-activated protein kinase pathway by ₂-adrenergic receptors expressed in fibroblasts. Journal of Biological Chemistry 268 22235–22238.[Abstract/Free Full Text]

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W & Lipman DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acid Research 25 3389–3402.[Abstract/Free Full Text]

Atkinson BN & Minneman KP 1992 Preferential desensitization of ß-versus ₂-adrenergic receptors accelerates loss of response to norepinephrine in primary glial cultures. Molecular Pharmacology 41 688–694.[Abstract]

Burden HW & Lawrence IE Jr 1973 Presence of biogenic amines in early rat development. American Journal of Anatomy 136 251–257.[CrossRef][Web of Science][Medline]

Buznikov GA, Shmukler YB & Lauder JM 1996 From oocyte to neuron: do neurotransmitters function in the same way throughout development? Cellular and Molecular Neurobiology 16 533–559.[CrossRef][Web of Science]

iko , Veselá J, Ilková G, Rehäk P, Czikková S & Koppel J 2005 Expression of beta adrenergic receptors in mouse oocytes and preimplantation embryos. Molecular Reproduction and Development 71 145–153.[CrossRef][Web of Science][Medline]

Cooper DMF 2003 Regulation and organization of adenylyl cyclases and cAMP. Biochemical Journal 375 517–529.[CrossRef][Web of Science][Medline]

Cussac D, Schaak S, Gales C, Flordellis Ch, Denis C & Paris H 2002 _2B-Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells. American Journal of Physiology. Renal Physiology 282 943–952.

Dickens CJ & Leese HJ 1994 The regulation of rabbit oviduct fluid formation. Journal of Reproduction and Fertility 100 577–581.[Abstract/Free Full Text]

Dickens CJ, Southgate J & Leese HJ 1993 Use of primary cultures of rabbit oviduct epithelial cells to study the ionic basis of tubal fluid formation. Journal of Reproduction and Fertility 98 603–610.[Abstract/Free Full Text]

Duzic E & Lanier SM 1992 Factors determining the specificity of signal transduction by guanine nucleotide–binding protein–coupled receptors III. Coupling of alpha adrenergic receptor subtypes in a cell type-specific manner. Journal of Biological Chemistry 267 24045–24052.[Abstract/Free Full Text]

Eason MG & Liggett SB 1995 Identification of a G_S coupling domain in the amino terminus of the third intracellular loop of the _2A-adrenergic receptor. Journal of Biological Chemistry 270 24753–24760.[Abstract/Free Full Text]

Einspanier R, Gabler C, Kettler A & Kloas W 1999 Characterization and localization of B₂-adrenergic receptors in the bovine oviduct: indication for progesterone-mediated expression. Endocrinology 140 2679–2684.[Abstract/Free Full Text]

Fernandez-Pardal J, Gimeno MF & Gimeno AL 1986 Catecholamines in sow graafian follicles at proestrus and at diestrus. Biology of Reproduction 34 439–445.[Abstract]

DeGraff JL, Gagnon AW, Benovic JL & Orsini MJ 1999 Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes. Journal of Biological Chemistry 274 11253–11259.[Abstract/Free Full Text]

Helm G, Owman C, Rosengren E & Sjoberg NO 1982 Regional and cyclic variations in catecholamine concentration of the human fallopian tube. Biology of Reproduction 26 553–558.[Abstract]

Herlenius E & Lagercrantz H 2001 Neurotransmitters and neuromodulators during early human development. Early Human Development 65 21–37.[CrossRef][Web of Science][Medline]

Higgins DG, Thompson JD & Gibson TJ 1996 Using CLUSTAL for multiple sequence alignments. Methods in Enzymology 266 383–402.[Web of Science][Medline]

Hutchinson DS, Bengtsson T, Evans BA & Summers RJ 2002 Mouse ß_3a-and ß_3b- adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways. British Journal of Pharmacology 135 1903–1914.[CrossRef][Web of Science][Medline]

Itoh MT, Ishizuka B, Kuribayashi Y, Abe Y & Sumi Y 2000 Noradrenaline concentrations in human preovulatory follicular fluid exceed those in peripheral plasma. Experimental and Clinical Endocrinology and Diabetes 108 506–509.[CrossRef]

Kannisto P, Ekblad E, Helm G, Owman C, Sjoberg NO, Stjernquist M, Sundler F & Walles B 1986 Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry. Histochemistry 86 25–34.[CrossRef][Web of Science][Medline]

Kennedy MF, Tutton PJ & Barkla DH 1983 Adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse. Clinical and Experimental Pharmacology and Physiology 10 577–586.[Web of Science][Medline]

Khatchadourian C, Menezo Y, Gerard M & Thibault C 1987 Catecholamines within the rabbit oviduct at fertilization time. Human Reproduction 2 1–5.[Medline]

Kim J, Eckhart AD, Eguchi S & Koch WJ 2002 Beta-adrenergic receptor-mediated DNA synthesis in cardiac fibroblasts is dependent on transactivation of the epidermal growth factor receptor and subsequent activation of extracellular signal-regulated kinases. Journal of Biological Chemistry 277 32116–32123.[Abstract/Free Full Text]

Kobayashi K, Morita S, Sawada H, Mizuguchi T, Yamada K, Nagatsu J, Hata T, Watanabe Y, Fujita K & Nagatsu T 1995 Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice. Journal of Biological Chemistry 270 27235–27243.[Abstract/Free Full Text]

Kotwica G, Kurowicka B, Franczak A, Grzegorzewski W, Wrobel M, Mlynarczuk J & Kotwica J 2003 The concentrations of catecholamines and oxytocin receptors in the oviduct and its contractile activity in cows during the estrous cycle. Theriogenology 15 953–964.

Lawits JA & Biggers JD 1993 Culture of preimplantation embryos. In Guide to Techniques in Mouse Development. Methods in Enzymology, pp 153–164. Eds PM Wassarman & ML DePhamphilis. San Diego: Academic Press.

Limon-Boulez I, Bouet-Alard R, Gettys TW, Lanier SM, Maltier JP & Legrand C 2001 Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells. Molecular Pharmacology 59 331–338.[Abstract/Free Full Text]

Lindquist JM, Fredriksson JM, Rehnmark S, Cannon B & Nedergaard J 2000 Beta 3- and alpha1- adrenergic Erk1/2 activation is Src- but not Gi- mediated in Brown adipocytes. Journal of Biological Chemistry 275 22670–22677.[Abstract/Free Full Text]

Liss B 2002 Improved quantitative real-time RT-PCR for expression profiling of individual cells. Nucleic Acids Research 30 e89.[Abstract/Free Full Text]

Mhaouty S, Cohen-Tannoudji J, Bouet-Alard R, Limon-Boulez I, Maltier JP & Legrand C 1995 Characteristics of the ₂/ß₂-adrenergic receptor-coupled adenylyl cyclase system in rat myometrium during pregnancy. Journal of Biological Chemistry 270 11012–11016.[Abstract/Free Full Text]

Mulder EHJ, Robles de Medina PG, Huizink AC, Van den Bergh BRH, Buitelaar JK & Visser GHA 2002 Prenatal maternal stress: effects on pregnancy and the (unborn) child. Early Human Development 70 3–14.[CrossRef][Web of Science][Medline]

Nepomnaschy PA, Welch KB, McConnell DS, Low BS, Strassmann BI & England BG 2006 Cortisol levels and very early pregnancy loss in humans. PNAS 103 3938–3942.[Abstract/Free Full Text]

Pendleton RG, Rasheed A, Roychowdhury R & Hillman R 1998 A new role for catecholamines: ontogenesis. Trends in Pharmacological Sciences 19 248–251.[CrossRef][Medline]

Pepperl DJ & Regan JW 1993 Selective coupling of alpha 2-adrenergic receptor subtypes to cyclic AMP-dependent reporter gene expression in transiently transfected JEG-3 cells. Molecular Pharmacology 44 802–809.[Abstract]

Pohjanoksa k, Jansson ChC, Luomala K, Marjamäki A, Savola JM & Scheinin M 1997 ₂-Adrenoceptor regulation of adenylyl cyclase in CHO cells: dependence on receptor density, receptor subtype and current activity of adenylyl cyclase. European Journal of Pharmacology 335 53–63.[CrossRef][Web of Science][Medline]

Pratt NC & Lisk RD 1989 Effects of social stress during early pregnancy on litter size and sex ratio in the golden hamster (Mesocricetus auratus). Journal of Reproduction and Fertility 87 763–769.[Abstract/Free Full Text]

Pullar CE & Isseroff RR 2003 PP2A activation by beta2-adrenergic receptor agonists: novel regulatory mechanism of keratinocyte migration. Journal of Biological Chemistry 278 22555–22562.[Abstract/Free Full Text]

Sadykova KA, Markova LN, Baikenova SD, Vsevolodov EB & Buznikov GA 1990 Biogenic monoamines in ovum cells and preimplantation embryos of mice. Biulleten’ Eksperimental’ noi Biologii i Meditsiny 109 577–578.[Medline]

Saunders C & Limbird LE 1999 Localization and trafficking of ₂-adrenergic receptor subtypes in cells and tissues. Pharmacology and Therapeutics 84 193–205.[CrossRef]

Schramm NL & Limbird LE 1999 Stimulation of mitogen-activated protein kinase by G protein – coupled alpha(2)-qdrenergic receptors does not require agonist-elicited endocytosis. Journal of Biological Chemistry 274 24935–24940.[Abstract/Free Full Text]

Seuwen K, Magnaldo I, Kobilka BK, Caron MG, Regan JW, Lefkowitz RJ & Pouyssegur J 1990 Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene. Cellular Regulation 1 445–451.

Shivachar AC & Eikenburg DC 1999 Differential effects of epinephrine and norepinephrine on cAMP response and G_i3 protein expression in cultured sympathetic neurons. Journal of Pharmacology and Experimental Therapeutics 291 258–264.[Abstract/Free Full Text]

Shizukuda Y & Buttrick PM 2002 Subtype specific roles of beta-adrenergic receptors in apoptosis of adult rat ventricular myocytes. Journal of Molecular and Cellular Cardiology 34 823–831.[CrossRef][Web of Science][Medline]

Slotkin TA, Yhang J, Dancel R, Garcia SJ, Willis C & Seidler FJ 2000 Beta-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells. Breast Cancer Research and Treatment 60 153–166.[CrossRef][Web of Science][Medline]

Temeles GL, Ram PT, Rothstein JL & Schultz RM 1994 Expression patterns of novel genes during mouse preimplantation embryogenesis. Molecular Reproduction and Development 37 121–129.[CrossRef][Web of Science][Medline]

Thomas SA, Matsumoto AM & Palmiter RD 1995 Noradrenaline is essential for mouse fetal development. Nature 374 643–646.[CrossRef][Medline]

Tolszczuk M & Pelletier G 1988 Autoradiographic localization of beta-adrenergic receptors in rat oviduct. Molecular and Cellular Endocrinology 60 95–99.[CrossRef][Web of Science][Medline]

Wang RX & Limbird LE 1997 Distribution of mRNA encoding three ₂-adrenergic receptor subtypes in the developing mouse embryo suggests a role for the _2A subtype in apoptosis. Molecular Pharmacology 52 1071–1080.[Abstract/Free Full Text]

Watson S & Arkinstall S 1994 Adrenaline and noradrenaline, The G-protein Linked Receptor Factsbook, London: Academic Press Limited, pp 32–55.

Way AL & Killian GJ 2002 Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine. Journal of Andrology 23 352–357.[Abstract/Free Full Text]

Way AL, Barbato GF & Killian GJ 2001 Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography. Life Sciences 70 567–576.[CrossRef][Web of Science][Medline]

Weiss ER, Maness P & Lauder JM 1998 Why do neurotransmitters act like growth factors? Perspectives on Developmental Neurobiology 5 323–335.[Web of Science][Medline]

Zhou QJ, Quaife CJ & Palmiter RD 1995 Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development. Nature 374 640–646.[CrossRef][Medline]

Zhu XH, He QL & Lin ZH 2003 Effects of catecholamines on human preadipocyte proliferation and differentiation. Zhonghua Zheng Xing Wai Ke Za Zhi 19 282–284.[Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cikos, S. Articles by Koppel, J. Search for Related Content PubMed PubMed Citation Articles by Cikos, S. Articles by Koppel, J.