Nature biotechnology & Cells alive!

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(by Johann Wolfgang von Goethe)

Terahertz radiation: biological objects under the submillimeter radiation.
actuality and novelty.

Studying an influence of biological objects illumination by impulsive lasers of low intensity in submillimeter ranges by mammal’s spermatozoa opens up new possibilities in the interpretation of qualitative and functional characteristics of cells. Such influence can be used: in the methods of cultivation in vitro for obtaining relatively pure cultures of stem cells of a pluripotential class of mammals’ marrow. According to the concept of tissue inhibitor-chalones (Bullough, 1975; Lee SJ, McPherron AC., 1999; Plikus MV, Mayer JA et al., 2008; Lander AD, Gokoffski KK et al., 2009), low differentiated cells are able to propagate continuously, but the process is restrained by the inhibitors released by differentiated cells. Increasing the activity of differentiation and the decrement of the mature cells quantity (Ol'shevskaia IuS, Kozlov AS, et al., 2009) – due to directional laser influence in the submillimeter range – promotes an accelerated maturation of ballast cells and a decrease of the proliferation inhibitors concentra-

tion. Therefore, it promotes an increase of the cytocinesis of the low differentiated cells that will lead to a rejuvenation of a monolayer cell composition with increasing by weighted rate of a part of stem cells of the pluripotential class; in IVF procedures by the treatment of infertile couples, by spermatozoa preparation for the oocytes fertilization; in different areas of the applied medicine for an acceleration of the regeneration cell processes, including cells growth, division, migration, as well as for a correction of the mechanisms of tissue proliferation regulation with normalization of the structure and tissue homeostasis dynamics; in diagnostics of hypofertility of men’s ambiguous genesis (by the correspondence of rates of a spermogram with reference values) according to distinctions in spermatozoa response to laser illumination; in agriculture by the use of cryopreserved sperm for the insemination of agricultural animals in order to increase sperm quality after storage and etc.

materials and methods.

Anative sheep ejaculate and its cryopreserved portions were chosen as a biological material. An electromagnetic influence in the submillimeter range on the biological object was carried out with the use of a gas-discharge laser with HCN working substance and the following basic technical characteristics: power 1,5 mW, exposures 1, 5 and 10 minutes, radiation frequency from 0,893 to 1,0 THz (with a wavelength about 0,337 mm). The native sheep ejaculate was transported in a thermostat at a temperature about 37 °C, and its cryopreserved portions – in a thermos on ice. Then the ejaculates were combined and they were poured 0,3 ml in 8 plastic capsule-like vessel with 5 mm diameter after mixing. A cryopreserved sperm in the form of frozen tablets was rapidly distributed at one tablet in similar capsule-like vessels (8 pieces) and then it was placed on ice again. The tests were pulled out the thermostat with given temperature (37 °C) directly before the influence by the laser. The cryopreserved samples were unfrozen directly before the influence of laser illumination and then they were put into a special thermal-resistant plate. The experimental tests of ejaculate were returned to the thermostat after influencing by the laser. Each test was irradiated in duplicates. A half of the experimental tests of the ejaculate were used for studying the intensity of chemiluminescence (ChL). The sample was diluted 1000 times for testing a concentration of the native sperm. The concentration of the sample made 1,8×10⁹ spermatozoa in 1,0 ml. Then it was counted the quantity of cells in 5 large squares of Goryaev chamber, placed diagonally (there were counted only spermatozoa whose heads were inside the square). One tablet must contain about 80×10⁶ spermatozoa in 1 ml to determine

the concentration of spermatozoa in cryopreserved sperm by standard methods. Therefore the samples of sperm were diluted 100 times and studied in Goryaev chamber. The concentration of spermatozoa in the samples is given below. All the samples were doubly washed with Tyrode buffer (pH 7,4, centrifugation at 1500 revolutions per minute, 5 minutes twice). After that the deposit of cells was resuspended by the same buffer in order to obtain a cell suspension followed by studying the rate of a formation of active oxygen radicals. The determination of the samples chemiluminescense was carried out on a chemiluminometer Lum-5773 (Russia), with the help of which there was done a quantitative count of the light emission, produced per 10 minutes in buffer-spermatozoa-luminol system. Obtained results were converted to the quantity of impulses, produced 20×10⁶ spermatozoa per 1 minute. For the morphological analysis spermatozoids were diluted with Earl’s solution so that the concentration of cells makes up about 20×10⁶ spermatozoa in 1 ml. Delivered ejaculate was divided into three portions: with samples staining by a fluorescent stain JC-1 (in concentration 1,0 mole×10^-3) with the exposition time about 10 minutes; with Rubenkov staining with cells fixation for the detection of anomalies of a morphologic construction; with staining by acridine orange to study the influence of different exposition of laser illumination on the continuity of nuclear chromatine. Then microscoping was carried out on a fluorescence microscope (Axio Imager M 2, objective 63×/1,4 Oil, Carl Zeiss, Germany) in order to study the influence of different laser exposure on spermatozoa mitochondria and the morphology of cells in a living culture (a drop under the cover glass without fixation).

results and deductions.

Physiologically healthy spermatozoa demonstrate the weak (often at the level of the background or lower) chemiluminescence (ChL). By increasing in the ejaculates the part of pathologically changed, damaged or cells with a weak mobility, the intensity of ChL significantly increases (1-3 times), that reflects the rate of production reactive oxygen radicals by cells. The hyperproduction of oxygen radicals can lead to a damage of spermatozoa structural components. These cells are especially sensitive to oxygen radicals because their plasmatic membrane contains a large quantity of polyunsaturated fatty acids, which easily enter into peroxidation lipids reaction (POL-process). And it decreases the continuity of membrane, influences the phosphorylation of axoneme proteins and leads to sperm cells immobilization. Thus, increasing ChL by spermatozoa is evidence of “oxidative stress” development. In studied tests at exposition for 1 minute ChL is observed at the level of background or less than zero, which gives rise to suppose the positive influence of submillimeter illumination on the biological object. On the contrary, the growth of the exposition up to 5 minutes and 10 minutes increases ChL by spermatozoa, that testifies about the development of "oxidative stress".

ChL of native sperm cells is less than cryopreserved ones, which can be connected with the fact that the remains of glycerin-lactose-yolk cryoprotector can also initiate ChL of luminol (C₈H₇N₃O₂). In native specimens of the ejaculate of sheep with JC-1 was observed until 5 % and in cryopreservation until 10 % spermatozoids with damage of acrosome and tail (control). Morphological analysis of the cells after irradiation with exposition 1 minute coincides with morphology of the control. More long expositions of the submillimeter radiation considerably increase number of the cells with damage of the cellular structure. Rubenkov morphology has shown there are less 5 % of damaged cells in samples with exposure 1 minute. On the contrary, in samples with exposure 5 and 10 minutes the morphology has become considerably worse and more 20 % of the cells were damaged. In the cryopreserved samples best morphology of the cells was observed (sharp contour of the acrosome) than in natives. The stain with acridine orange gives the morphology without features (the spermatozoids heads are easily visible, but of damaged membranes and violation of the condensation of the nuclear chromatin was not detected).

strategics and prospects.

By the end of 2009-2010 several experiments with the cellular material of the sheep ejaculate are planned in order to continue the study of the influence of electromagnetic waves of the submillimeter range on the biological object in a modeled mode with modula-

tion frequency from 1 to 35 Hz are planned.
The obtained results permit to approximate to the interpretation of the possibility of the regulation of cellular proliferation mechanisms and the provision of cellular homeostasis as a factor of tissue stability.

Native ejaculate. The morphology without features (control). The morphology without features.

Photo. 1. Native ejaculate. Control. The morphology without features. (by Rubenkov)

Cryopreserved ejaculate. The morphology without features (control). The morphology without features.

Photo. 2. Cryopreserved ejaculate. Control. The morphology without features. (by Rubenkov)

Photo. 3. Native ejaculate. Exposure 1 minute. The morphology without features, less 5 % of damaged cells. (by Rubenkov)

Photo. 4. Cryopreserved ejaculate. Exposure 1 minute. The morphology without features, less 5 % of damaged cells. (by Rubenkov)

Photo. 5. Native ejaculate. Exposure 5 minutes. More 20 % of the cells were damaged. (by Rubenkov)

Native ejaculate. Exposure 1 minute. The morphology without features. Violation of the condensation of the nuclear chromatin was not detected.

Photo. 6. Native ejaculate. Exposure 1 minute. The morphology without features. Violation of the condensation of the nuclear chromatin was not detected. (acridine orange)