ISSN 00124966, Doklady Biological Sciences, 2012, Vol. 442, pp. 38–41. © Pleiades Publishing, Ltd., 2012. Original Russian Text © N.Yu. Sakharova, L.N. Markova, A.A. Smirnov, E.F. Vikhlyantseva, L.A. Fialkovskaya, V.V. Bezuglov, 2012, published in Doklady Akademii Nauk, 2012, Vol. 442, No. 4, pp. 567–569.
GENERAL BIOLOGY
Effect of Docosahexaenoyl Dopamine on the in Vitro Development of Early Mouse Embryos N. Yu. Sakharovaa, L. N. Markovab, A. A. Smirnova, E. F. Vikhlyantsevaa, L. A. Fialkovskayac, and V. V. Bezuglovd Presented by Academician A.I. Miroshnikov September 10, 2011 Received September 29, 2011
DOI: 10.1134/S0012496612010115
Involvement of neurotransmitters in early (preneu ral) embryogenesis was mainly studied on embryos of marine invertebrates and low vertebrates [1, 2]. As for mammals, it was demonstrated that there are struc tures in preimplantation mouse embryos that are sen sible to antagonists of serotonin, adrenalin, and ace tylcholine [3–6], and cAMP is involved in the func tional activity of these substances [7]. Not only their receptors, but also other components of transmitter systems (specific transporter proteins, metabolic enzymes) were detected in oocytes and embryos of mammals [8, 9]. The affect of dopamine is the least studied, since it is extremely unstable in the incuba tion medium. Due to the creation of artificially func tionalized amides of polyene fatty acids with biogenic monoamides, it became possible to use amides of dopamine with fatty acids instead of dopamine itself. These substances were the most suitable for studying the role of classical neurotransmitters in biological systems, since they easily penetrate through the cell membrane and are stable in the incubation medium. Their action is also of independent interest, since they have their own biological activity not limited to the activity of their biogenic amines and fatty acids [10, 11].
ment of preimplantation mouse embryos. Embryos of SHK mice isolated at the stages of two, four, and eight blastomeres were placed into M16 medium (Sigma) supplemented with DHADA at a concentration of 2 µM and cultured in a CO2 incubator (Binder, Ger many) for two to three days until embryos of the con trol groups reach the final stage of preimplantation development (blastocyst) (Fig. 1a). Observation of the development was performed every 24 h using a Leica DM JL stereoscopic microscope (Germany); photo
(a)
(b)
(c)
(d)
We studied the effect of dopamine conjugate with docosahexaenoic acid (DHADA) on the develop
a
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia b Koltsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia c Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia d Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. MiklukhoMaklaya 16/10, Moscow, 117997 Russia
Fig. 1. (a) The stage of blastocyst, the final stage of the development of the control embryos in all series of the experiment; (b, c, d) embryos treated with DHADA: (b) arrest of development of a twocell embryo at the stage of the cleavage; (c) arrest of development of a fourcell embryo at the stage of morula; (d) arrest of development of an eightcell embryo at the stage of early blastocyst.
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EFFECT OF DOCOSAHEXAENOYL DOPAMINE
39
Influence of DHADA on the development of early mouse embryos in vitro 24 h stages of the development
48 h
% of embryos control
experiment
stages of the development
72 h
% of embryos control
experiment
stage of the development
% of embryos control
experiment
Twocell embryos (control, 29 embryos; experiment, 30 embryos) 2 blastomeres 27.8 ± 9.8
8.4 ± 5.1 2 blastomeres 14.0 ± 9.8
4 blastomeres 68.0 ± 6.2
74.5 ± 8.3 4 blastomeres 12.4 ± 8.1
8 blastomeres Embryos with abnormal structure
10.2 ± 4.5 14.8 ± 8.0 8 blastomeres 10.6 ± 4.8 0
2.2 ± 2.2 8 compact blastomeres
0
Arrest of cleavage
41.5 ± 17.7 Morula
27.4 ± 14.0 28.6 ± 17.6
33.5 ± 19.2 Blastocyst
48.3 ± 17.2 17.2 ± 10.2
60.8 ± 13.8 *14.3 ± 14.3 Embryos 12.2 ± 9.7 23.5 ± 10.6 with abnormal structure
Morula
11.1 ± 11.1
0
0
10.7 ± 6.9
Embryos with abnormal structure
Fourcell embryos (control, 23 embryos; experiment, 26 embryos) 4 blastomeres
8.3 ± 6.0
6.6 ± 4.4 8 compact blastomeres
8 blastomeres
15.3 ± 11.1 23.8 ± 8.5 Morula
8 compact blastomeres
38.0 ± 14.1 69.6 ± 10.6 Blastocyst
Morula
35.6 ± 14.2
0*
2.8 ± 2.8
0
0
0
Blastocyst Embryos with abnormal structure
0
4.2 ± 4.2
5.6 ± 5.6
31.6 ± 18.1
88.2 ± 7.0 *38.8 ± 15.2
Embryos with abnormal structure
5.6 ± 5.6
25.5 ± 11.6
Eightcell embryos (control, 31 embryos; experiment, 34 embryos) 8 blastomeres
0
0
8 blastomeres
0
0
8 compact blastomeres
16.1 ± 8.3 34.3 ± 14.6 8 compact blastomeres
4.1 ± 4.1
0
Morula
50.4 ± 12.9 33.1 ± 14.6 Morula
4.8 ± 4.8
32.1 ± 17.9
Blastocyst
31.4 ± 16.2
0
Blastocyst
86.2 ± 7.0 *16.1 ± 9.2
Embryos 2.04 ± 2.04 32.6 ± 15.2 Embryos with abnormal with abnormal structure structure
4.9 ± 3.2 *51.8 ± 15.7
* Significant differences from the control group, p < 0.05. DOKLADY BIOLOGICAL SCIENCES
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SAKHAROVA et al.
graphs were made using an Axiovert 200 Zeiss inverted microscope (Germany).
tomeres. However, the subsequent compaction is dis turbed.
The state of the embryos was assessed from their morphology [12]. The numbers of normally develop ing, abnormal, and nondeveloping embryos were detected. A total of 173 embryos were used in experi ments. The results obtained were processed using Stu dent’s t test and a modified Microsoft Excel software to calculate the standard error.
Fourcell embryos under the experimental condi tions successfully pass a single division and start the compaction. At the stage of eight compact blas tomeres, a delay in the development occurs, and embryos progress to the stage of morula more slowly than in the control group. Subsequently, after 48 h, disturbances in the processes of the blastocyst devel opment (which result in the abnormal structure of most embryos) are expressed.
The results (table) obtained during the cultivation of embryos isolated at the stage of two blastomeres demonstrated that, after 24 h, most embryos in both control and experimental groups reached the stage of four to eight blastomeres. After 48 h, most control embryos were at the stage of eight compact blas tomeres, while most experimental embryos were at the stage of four to eight blastomeres. After 72 h, more than half the control embryos reached the final stage of preimplantation development (blastocyst). In the experimental group, most embryos were at different stages of cleavage, and many of them had an abnormal structure (Fig. 1b). Most embryos that had developed to the stage of blastocyst were also characterized by disturbances in the structure of the inner cell mass (ICM) and trophoblast. In the group of embryos isolated at the stage of four blastomeres, most control and experimental embryos were at the stage of eight compact blastomeres after 24 h of culturing. Some control embryos were at the next stage (multicellular morula). No such morulae were found among the experimental embryos. In the experimental group, half as many embryos reached the stage of blastocyst within 48 h compared to the control group. The experimental embryos were mostly clusters of large cells; i.e., they were apparently abnormal morulae (Fig. 1c). The culturing of embryos isolated at the stage of eight blastomeres demonstrated that, after 24 h, most control embryos were at the stage of morula or blasto cyst, while most experimental embryos were at the stages of eight compact blastomeres and morula. A large amount of abnormal embryos were observed in the experimental group. After 48 h, most embryos reached the stage of blastocyst in the control group, while only a small proportion reached this stage in the experimental group. In most experimental embryos, signs of abnormal structure were found (Fig. 1d). Comparison of the results obtained in the experi ments on the influence of DHADA on the embryos at different stages of cleavage allows us to detect the characteristics of its effect on the early mouse develop ment in vitro. DHADA does not inhibit the continuation of the cleavage of twocell embryos when they pass two rounds of division and reache the stage of eight blas
Experimental eightcell embryos during the first 24 h of the cultivation successfully develop to the stage of morula; however, they exhibit a decelerated transi tion to the blastocyst stage. The observed considerable percentage of embryos with abnormal structure indi cates the disturbance in the development of blastocyst. This is confirmed by the results of observation after 48 h of culturing: a much smaller amount of embryos reaches the stage of blastocyst than in the control group. Thus, the results presented demonstrate that DHADA influences the embryos at all stages studied. Thus, DHADA did not stop the cleavage of twocell embryos, but it disturbed the subsequent compaction at the stage of eight blastomeres. DHADA did not interrupt the division of fourcell embryos and subse quent compaction at the stage of eight blastomers, but it inhibited the subsequent development of a multicel lular morula. Eightcell embryos in the presence of DHADA pass through compaction and the genera tion of the morula, but the disturbance in the forma tion of the blastocyst occurs. These data demonstrate that DHADA does not disturb the current stage, but it inhibits the transition to the next stage. Earlier, we studied the effect of dopamine conjugate with another fatty acid (arachidonic) (AADA) [13]. As opposed to DHADA, AADA, when applied at a similar concen tration (2 µM), considerably inhibited the cleavage of twocell embryos. This difference may have been due to the action of both the molecule as a whole and its individual components. There is an assumption that 3hydroxytyramides (which include DHADA and AADA) are universal endogenous regulators of early embryogenesis [11, 14]. The data on the effect of DHADA on mouse embryos agree with the concept that early develop ment of mammals consists of consecutive stages; their triggering is determined by factors that appear at a cer tain moment at an earlier stage [15]. Apparently, DHADA influences the processes that determine the next stage of the development but does not inhibit the completion of the current morphogenetic events. DOKLADY BIOLOGICAL SCIENCES
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ACKNOWLEDGMENTS This study was supported by the Russian Founda tion for Basic Research, project no. 110401469a. REFERENCES 1. Buznikov, G.A., Neurotransmitters in Embryogenesis, Chur: Harwood Academic, 1990. 2. Buznikov, G.A., Ontogenez, 2007, vol. 38, no. 4, pp. 262–270. 3. Burden, H.W. and Lawrence, J.E., Am. J. Anat., 1973, vol. 136, pp. 251–257. 4. Sadykova, K.A., Markova, L.N., Baikenova, S.D., et al., Byull. Eksp. Biol. Med., 1990, vol. 109, pp. 577– 578 5. Sakharova, N.Yu., Markova, L.N., Sadykova, K.A., et al., Ontogenez, 1995, vol. 26, pp. 48–53. 6. Markova, L.N., Sadykova, K.A., and Sakharova, N.Yu., Zh. Evol. Biokhim. Fiziol., 1990, vol. 26, pp. 726–732.
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7. Sadykova, K.A., Sakharova, N.Yu., and Markova, L.N., Ontogenez, 1992, vol. 23, no. 4, pp. 379–384. 8. Mayerhofer, A., Smith, G. D., Danilchik, M., et al., Proc. Nat. Acad. Sci. U.S.A., 1998, vol. 95, pp. 10 990– 10 995. 9. Dube, F. and Amireault, P., Life Sci., 2007, vol. 81, pp. 1627–1637. 10. Bezuglov, V.V., Manevich, E.M., Archakov, A.V., et al., Bioorg. Khim., 1997, vol. 23, pp. 211–220. 11. Buznikov, G.A. and Bezuglov, V.V., Ross. Fiziol. Zh. im. I.M. Sechenova, 2000, vol. 86, no. 9, pp. 1093–1108. 12. Pratt, Kh., Biologiya razvitiya mlekopitayushchikh. Metody (Biology of Mammalian Development: Meth ods), Moscow: Mir, 1990. 13. Markova, L.N., Sakharova, N.Yu., and Bezuglov, V.V., Ontogenez, 2000, vol. 31, no. 1, pp. 32–39. 14. Akimov, M.G., Gretskaya, K.V., Shevchenko, V.P., et al., Bioorg. Khim., 2007, vol. 33, pp. 648–652. 15. Evsikov, A.V., Ontogenez, 2000, vol. 31, no. 3, pp. 178– 191.
