Процессы, происходящие при созревании яйцеклеток животных, - одна из ключевых проблем биологии развития. являются важным компонентом клеток животных. Роль жирных кислот в процессах созревания, оплодотворения и развития яйцеклеток млекопитающих изучена недостаточно. Ранее авторами было показано, что способность морфологически нормальных ооцитов достигать in vitro стадии метафаза II не зависит от морфофункционального состояния яичника коров. Неизвестно, однако, влияет ли исходное морфофункциональное состояние яичника на биохимическиe процессы в ооцитах, в частности, на их химический состав. Для определения жирнокислотного состава общих липидов ооцитов в связи с процессом ядерного созревания в данной работе объединяли данные по всем пяти морфофункциональным состояниям яичников у коров. Показано, что морфофункциональное состояние яичников существенно не влияло на жирнокислотный состав общих липидов ооцитов. Установлено, что жирные кислоты в основном представлены ненасыщенными формами, как в ооцитах с первым направительным тельцем, так и без него (76,7 и 83,1% cоответственно). Ооциты с первым направительным тельцем содержали существенно больше пальмитиновой (12,0 vs 7,3%), стеариновой (7,37 vs 5.81%) и олеиновой кислот (6.00 vs 4,63%) и меньше линолевой (37,1 vs 39,5%), линоленовой (27,0 vs 31, 3%) и арахидоновой кислот (6,33 vs 7,55%) по сравнению с ооцитами без первого направительного тельца. Выявлены устойчивые изменения в жирнокислотном составе общих липидов ооцитов крупного рогатого скота в связи с процессом ядерного созревания in vitro. Это позволяет говорить о наличии некоего универсального механизма, регулирующего процесс ядерного созревания. ABSTRACT. The processes occurring during the maturation of animal oocytes is one of the key problems in developmental biology. are an essential component of animal cells. The role of fatty acids in the processes of maturation, fertilization and development of mammalian oocytes has been insufficiently studied. Previously, the authors showed that the ability of morphologically normal oocytes to reach the metaphase II stage in vitro does not depend on the morphofunctional state of the cow ovary. It is not known, however, whether the initial morphological and functional state of the ovary affects biochemical processes in oocytes, in particular, their chemical composition. To determine the fatty acid composition of the total lipids of oocytes in connection with the process of nuclear maturation, this study combined data on all five morphofunctional states of the ovaries in cows. It was shown that the morphofunctional state of the ovaries did not significantly affect the fatty acid composition of the total lipids of oocytes. It was found that fatty acids are mainly represented by unsaturated forms, both in oocytes with and without the first targeting body (76.7 and 83.1%, respectively). Oocytes with the first targeting body contained significantly more palmitic (12.0 vs 7.3%), stearic (7.37 vs 5.81%) and oleic acids (6.00 vs 4.63%) and less linoleic (37.1 vs 39, 5%), linolenic (27.0 vs 31.3%) and arachidonic acids (6.33 vs 7.55%) compared to oocytes without the first targeting body. Stable changes in the fatty acid composition of the total lipids of cattle oocytes were revealed in connection with the process of nuclear maturation in vitro. This allows us to speak about the presence of a certain universal mechanism that regulates the process of nuclear maturation.

The opportunity to create a chemically defined system for bovine sperm in vitro capacitation has been performed. 100 µM dibutyryl cyclic adenosine monophosphate (dbcAMP) used as a capacitating agent instead of heparin 10 µg/mL solution was administered in a Tyrode’s protein-free medium for fertilization. In addition, the fertilization medium did not contain glucose or any sperm-activating additives. It has been proven for the first time that spermatozoa capacitated in the in vitro protein-free medium with the use of dbcAMP only are capable of highly efficient in vitro fertilization of the bovine oocytes, which were matured outside the body in a protein-free medium as well. Embryos developed to the 4- to 8-cell stage are chosen as criteria for normal fertilization.

Цель работы - изучить эффективность созревания ооцитов крупного рогатого скота in vitro в безбелковой среде MEM-α, дополненной двумя различными препаратами фолликулостимулирующего гормона (полученного из свиных гипофизов и рекомбинантного человеческого ФСГ). Критерием успешного созревания считали способность ооцитов после оплодотворения развиваться до стадии морулы-бластоцисты и бластоцисты. Показано, что ооциты, созревавшие в присутствии рекомбинантного человеческого гормона, лучше развивались до стадии морулы-бластоцисты (в процентах от дробившихся эмбрионов), и этот эффект сохранялся при развитии до стадии бластоцисты. Заключили, что рекомбинантный ФСГ человека может использоваться для созревания ооцитов крупного рогатого скота in vitro наравне с “классическим” гормоном, полученным из свиных гипофизов. The aim was to study the efficiency of maturation of bovine oocytes in vitro in a protein-free medium MEM-alfa supplemented by two various preparations of follicle stimulating hormone, isolated from pig pituitary glands and recombinant. The development of in vitro maturated and fertilized oocytes to morula-blastocyst stage and to blastocyst stage was considered as criterion of successful maturation. It has been shown that oocytes matured in the presence of recombinant human hormone developed better to the morula-blastocyst stage (as a percentage of divided embryos), and this effect was maintained during development to the blastocyst stage. It was concluded that recombinant human FSH can be used for the maturation of cattle oocytes in vitro along with the “classical” hormone obtained from porcine pituitary glands.

Аннотация - Представлены результаты исследования структурирования поверхности стекла с субдифракционным пространственным разрешением с использованием фемтосекундного лазерного излучения, сфокусированного микронным диэлектрическим шариком, покрытым наночастицами золота. Показано, что при использовании излучения с длиной волны 780 нм достигается размер отверстия 110 нм. Предложен механизм абляции. Методика открывает новые возможности для создания микрофлюидных и сенсорных устройств в технологии Lab-on-Chip для обнаружения токсичных и взрывоопасных веществ. Abstract - The results for nanostructuring glass surface with subdiffraction spatial resolution using femtosecond laser radiation focused by core/shell type Au/SiO2 microspheres are presented. A spatial resolution value of 110 nm has been achieved by applying laser radiation at a wavelength of 780 nm. An appropriate mechanism for glass ablation is proposed. The technique opens up new opportunities for creating microfluidic and sensor devices based on Lab-on-Chip technology which can be successfully applied for detecting toxic and explosive substances.

Changes of chemical composition and optical properties due to the femtosecond laser action on the biomaterials was characterized by multiphoton luminescence, femtosecond CARS microscopy techniques, atomic force microscopy and time of flight secondary ions mass spectroscopy. The survival and ability to development of mammalian embryos has been studied.

The effects of hypoxanthine (HX) at low concentrations on the ability of bovine oocyte to mature to metaphase 2 (M2) and to fertilize in vitro have been studied. It is proved that exogenous HX even at a low concentration, which was introduced into the HX-free and protein-free alpha-MEM medium supplemented with 1 μg/mL follicle-stimulating hormone, can significantly decrease the proportions of both the oocytes the matured to stage M2 and the penetrated and normally fertilized oocytes. It is indicated that the data may be used in order to develop commercial media for in vitro maturation of oocytes of mammalia, including humans.

The aim of the investigation was to determine a noninvasive range of near-infrared femtosecond laser pulse exposure for mouse oocyte intracellular nanosurgery and to estimate oocyte developmental competence in vitro depending on pulse energy and total exposure time.Materials and methods. The model object was a preovulatory germinal vesicle mouse oocyte. Femtosecond laser radiation with a central wavelength of 780 nm was utilized in two modes: 1) single pulses with 30 fs duration and pulse energy up to 100 nJ; 2) trains of pulses with 100 fs duration, 80 MHz repetition rate (12.5 ns between pulses) and pulse energy from 0.5 to 2 nJ; total duration of the pulse trains ranged from 15 to 60 ms. Radiation was focused by a microscope objective lens (60x, 0.7 NA). Oocytes were cultured to the metaphase of the second meiotic division, which was used as invasiveness criteria. This stage was detected by polar bodies and metaphase plate formation.Results. The threshold of invasiveness was determined for a single femtosecond pulse exposure. Cytoplasmic membrane rupture and cell disruption occurred under the exposure of a single 30 fs pulse with 100 nJ energy. The cytoplasm and nucleus exposure to femtosecond pulse trains did not induce permanent damage to the oocytes. The rate of gas-vapor bubble formation was measured after the action of pulse trains on the cytoplasm and nucleus chromatin of mammalian oocytes. The pulse energy of 1 nJ is a threshold value for bubble formation in the cytoplasm, and 2 nJ in the nucleus. Exposure to laser pulse trains with 80 MHz repetition rate and 100 fs duration didn't change oocyte ability to achieve metaphase II stage within the studied range of pulse energies and pulse train durations.Conclusion. Surgery with femtosecond pulse trains at 80 MHz repetition rate and pulse energy not more than twice as high as the threshold of cavitation or vapor bubble formation does not affect oocyte development to the metaphase II stage. Femtosecond laser nanosurgery can be regarded as a minimally invasive method of intracellular surgery within the investigated range of pulse energies and exposure times.

The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.

Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60 %. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated.

The efficiency of cattle oocyte maturation in vitro was studied in protein-free MEM-α with hormones and in completely definite culture medium without hormones. Oocyte capacity to develop after fertilization to the morula/blastocyst and blastocyst stages served as a criterion of effective maturation. The increase in follicle-stimulating hormone concentration in the medium by one or two orders of magnitude in comparison with the “standard” level of 1 μg/ml deteriorated the development of embryos to the preimplantation stages. Serum gonadotropin from pregnant mares worked similarly as follicle-stimulating hormone. Oocytes that underwent maturation without hormones developed to the blastocyst stage, though the percentage of dividing embryos was significantly less and there was a trend to worse development of the embryos to the preimplantation stages.

Описано получение химерных бластоцист мыши методами лазерной нанохирургии без использования какой-либо другой дополнительной техники. Для проведения экспериментов был разработан и сконструирован специальный лазерный микроманипулятор. В экспериментах использовали эмбриональные стволовые клетки (ЭСК) мыши, трансформированные вектором pEF-GFP, кодирующим флуоресцирующий зеленый белок. ЭСК с помощью лазерного микроманипулятора вводили в перивителлиновое пространство мышиных эмбрионов на стадии 8-и клеток. Оперированные эмбрионы культивировали in vitro до стадии вылупления из zona pellucida. Наличие флуоресценции и ее точную локализацию регистрировали на конфокальном микроскопе. Впервые было продемонстрировано, что включения введенных лазерным микроманипулятором ЭСК, обнаруживаются как во внутренней клеточной массе (ВКМ), так и в трофэктодерме химерной бластоцисты. Технология проведения нанохирургических операций на ранних доимплантационных эмбрионах млекопитающих с помощью лазерной техники открывает большие возможности не только для решения фундаментальных задач экспериментальной эмбриологии млекопитающих, но эта технология также может быть использована как перспективный метод получения химерных и трансгенных животных с заданным экспериментаторами генотипом.

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods with-out using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.

The application of etoposide for chemical enucleation of rat oocytes was tested. The reconstruction efficiency after chemical and mechanical enucleation was comparatively analyzed. The obtained data indicate similar viability of reconstructed rat embryos irrespective of the enucleation technique.

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.

We studied the capacity of cattle oocytes taken from ovaries with different morphofunctional state for development to metaphase II in vitro . A classification of ovaries has been proposed according to their morphofunctional state: (1) ovaries with a yellow body from the last cycle, without dominating follicle, with many follicles of varying diameter; (2) ovaries with a yellow body from the last cycle, with dominating follicle (from 10 mm in diameter); (3) ovaries with a large functioning yellow body and follicles of varying diameter; (4) ovaries with a follicular cystoid formation (more than 25 mm in diameter); (5) ovaries with a yellow body from past cycles and small (1–2 mm) follicles, supposedly with a weakened hormonal function. It was shown that the morphofunctional state of ovaries determined the total number of oocytes isolated from an ovary and number of morphologically normal oocytes feasible for cultivation. At the same time, no reliable differences in the capacity for extrusion of the first polar body between the oocytes from the ovaries of different types were found in the experiments on in vitro oocytes maturation. Since the coefficient of correlation between the extrusion of the first polar body and maturation to metaphase II was in 0.95, there is every reason to believe that the capacity for development to metaphase II does not depend on the morphofunctional state of ovaries.