Infradian about 4-day fluctuations were found in many biological parameters: concentrations of melatonin, glucocorticoid and sex hormones [1-6], motor activity [7], immune system [8, 9], mitotic activity of cells [10, 11]. Based on the facts that 1) 4-day rhythms are preserved during the removal both of the testes and adrenal glands [12], and 2) hibernating hedgehogs with reduced hormonal activity had the stable rhythms of spontaneous short-term arousals [13], it can be argued that the formation of this infradian rhythm is not associated with hormonal regulation. It is interesting that the removal of the pineal gland, which is involved in the formation of circadian rhythms [14], also does not affect the period and phase of the 4-day rhythm of mitotic activity of the rat esophagus epithelium [7]. Probably, the 4-day rhythm is determined by the activation of the sympathetic nervous system, which is indicated by the presence of this rhythm in the dynamics of epinephrine excretion in the urine [2], as well as the whole complex of signs associated with an increase in motor activity, the release of glucocorticoid hormones, suppression of immune reactions, a decrease in proliferative cell activity.
The mitotic activity of epithelial cells is the convenient model of the stable manifestation of 4-day rhythm [11]. However, the formation mechanisms of this rhythm have not been established yet. In vivo studies do not allow to exclude the influence on the mitotic activity of cells of humoral factors of the body internal environment, that have a 4-day rhythm determined by the activation of the sympathetic nervous system. Based on this hypothesis, the 4-day rhythm of mitotic activity in an isolated cell culture should be absent. However, studying the daily dynamics of the proliferative activity of the culture of embryonic fibroblast-like cells in the logarithmic growth phase, we revealed a 4-day rhythm that persists for at least three weeks of cultivation [15].
There are three possible variants of this rhythm’s origin: 1) completely endogenous, self-sustaining on the basis of molecular genetic interactions, and, therefore, such a rhythm will not be synchronous in different cell cultures; 2) completely exogenous, caused by the influence of an external environmental factor; 3) endogenous, but having an external synchronizer, and, therefore, these two rhythms will be synchronous.
To determine the possible origin of the 4-day rhythm of the proliferative activity of fibroblast-like cells, it is necessary to establish:
1) are the manifestations of the 4-day rhythm of proliferative activity in cultures obtained on different days from embryos of the same age synchronous?
2) does the phase of the rhythm in cell culture comparable with that in vivo? Under in vivo conditions, the phase of the infradian rhythm of the motor activity of animals is observed in antiphase to the 4-day rhythm of mitotic activity (mitotic index) of the esophageal epithelium [7].
3) will freezing of cells for a period not multiple of 4 days cause a phase shift of the investigated infradian rhythm relative to the intact culture?
However, immediately after thawing, embryonic fibroblast-like cells do not begin to divide intensively. It is a considerable time period between the isolation of the culture and the beginning of the experiment, during which the cultures can synchronize their endogenous rhythms. Therefore, at the final stage of our research, we studied the changes in the proliferative activity of the fiboroblast-like cells mice culture L-929, which has a high proliferative potential.
The aim of the 4th point of the study was: to establish the exogenous/endogenous nature of about 4 days of the rhythm of proliferative activity of fibroblast-like L-929 cells by daily thawing of same frozen samples of the same culture. If, after defrosting, the rhythm is immediately observed in the phase, as if it were not stopped, then we can suggest its exogenous origin, but if the restoration of the rhythm phase is observed gradually, then this is an endogenous rhythm that has an external synchronizer.