Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro.

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.

Effects of hormones on in vitro maturation of cattle oocytes.

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.

[Production of reconstructed two-cell rat embryos after chemical inactivation of chromosomes in MII oocytes by etoposide].

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.

[In vitro fertilization of bovine oocytes in in-vitro protein-free culture system].

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.

[In vitro maturation of cattle oocytes taken from ovaries with different morphofunctional states]. / Sozrevanie in vitro ootsitov krupnogo rogatogo skota, vziatykh iz iaichnikov razlichnogo morfofunktsional'nogo sostoianiia.

We studied the capacity of cattle oocytes taken from ovaries with different morphofunctional state for development to metaphase 2 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 2 was in 0.95, there is every reason to believe that the capacity for development to metaphase 2 does not depend on the morphofunctional state of ovaries.

[In vitro development of murine embryos using different types of microinjections]. / Razvitie myshinykh émbrionov in vitro pri razlichnykh variantakh mikroin''ektsii.

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA x x C57BL)F1. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.

[Investigation of the effect of various buffer solutions used for microinjection of gene-engineering constructs on preimplantation development of mouse embryos]. / Izuchenie vliianiia razlichnykh rastvorov, ispol'zuemykh pri mikroin''ektsii genno-inzhenernykh konstruktsii, na doimplantatsionnoe razvitie myshinykh émbrionov.

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl2 and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.

[Fertilization and subsequent development of cattle oocytes after reduced incubation with spermatozoa]. / Oplodotvorenie i posleduiushchee razvitie ootsitov krupnogo rogatogo skota posle cokrashchennoi inkubatsii so spermatozoidami.

We studied the influence of the duration of joint incubation of cattle oocytes and spermatozoa (18 versus 1 h) on fertilization, cleavage, and embryonic development in vitro until the blastocyst stage. Spermatozoa of a bull of the Britanofrizskaya breed were used in the experiments. It was shown in the first experimental series that after a long-term incubation with the spermatozoa, the percentage of penetrated eggs increased: 71.7% and 56.0% (p < 0.05) after 18-hour and 1-hour incubation, respectively. However, no differences were found in the number of normally fertilized eggs: 46.5 and 39.0%, respectively. In the second experimental series, no significant differences were found in either the number of cleaving embryos (41.2 and 32.2%, respectively) or the capacity of cleaving embryos to develop in vitro until the blastocyst stage (21.7 and 15.8%, respectively). Thus, reduction in the time of joint incubation of cattle gametes upon in vitro fertilization to 1 h did not reduce the number of normally fertilized eggs and did not affect their capacity for subsequent in vitro development.

[The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro]. / Vliianie sostava kul'turnykh sred na sozrevanie ootsitov i razvitie émbrionov krupnogo rogatogo skota in vitro.

We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA. In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium. Meiosis was not resumed in Ham's F-12. Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%). The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively). In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization. The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively). However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).

[The biopsy of preimplantation embryos obtained from donor pigs with follicle-stimulating hormone-induced superovulation]. / Biopsiia doimplantatsionnykh émbrionov, poluchennykh ot svinei-donorov s vyzvannoi follikulostimuliruiushchim gormonom superovuliatsiei.

We have examined possible use of the follicle-stimulating hormone (FSH) for the induction of superovulation in pigs and studied the effect of biopsy of preimplantation pig embryos on their survival in vitro. Superovulation was induced by injecting FSH twice daily over a period of four days for a total dose of 25 units. per animal. Pigs receiving pregnant mare serum gonadotropin (PMSG) according to the standard scheme served as the control. The experiments demonstrated that FSH produces significantly better estrus figures as compared with PMSG (100% and 60%, respectively; p < 0.05). The mean number of ovulations per donor was also better in the FSH group, i.e., 36.5 as compared with 17.3 in the control group (p < 0.05). The mean number of embryos obtained from one donor was higher in FSH treated animals as compared with the animals that received PMSG (29.2 vs. 19.3), however, this difference was not statistically significant. Biopsy of preimplantation pig embryos was conducted by a "manual" technique without a micromanipulator; middle blastocysts collected at days 5-6 after insemination were the most convenient for this operation. Embryos at these developmental stages contained a well developed inner cell mass, which allows separation of trophoectodermal cells only, thus minimizing damage to the embryo. The number of cells in dissected fragments did not depend on the stage of development, and even smallest fragments (4 blastomeres) were sufficient for genome analysis with the aid of PCR. The survival of embryos in vitro after biopsy practically did not differ from that of control intact embryos. Thus, we demonstrated the effective use of FSH for the induction of superovulation in pigs; we also determined the developmental stages which are most convenient for conducting biopsies and developed a technique for preimplantation biopsy, which allows genome analysis of embryos without any decrease of their survival in vitro.

[The creation of genetic cryobanks and the use of the methods of developmental biology as a means for preserving rare animal species. III. The outlook, problems and limits: selection, modifications and mutations]. / Sozdanie geneticheskikh kriobankov i ispol'zovanie metodov biologii razvitiia kak sposob sokhraneniia redkikh vidov zhivotnykh. III. Perspektivy, problemy i ogranicheniia: selektsiia, modifikatsii i mutatsii.

A review of our own and literature data about perspectives, problems and limitations in the use of cryoconservation techniques and methods of developmental biology for the conservation of genetic resources is presented. It is demonstrated that the use of these methods may result in the selection and possibly leads to modifications and mutations in germ cells and embryos and in this way may change the genetic structure of restored populations as compared with the original ones. The need to improve current techniques of cryoconservation and accompanying biotechnological procedures in order to abolish these undesirable genetic effects is emphasized.

[Ultrafast freezing of the embryos of outbred and inbred mouse strains]. / Sverkhbystroe zamorazhivanie émbrionov myshei autbrednykh i inbrednykh linii.

Survival of 8-cell mouse embryos of inbred strains C57B1 and DBA and outbred strain NMRI was studied after ultrafast freezing. The embryos were placed in 10% glycerol for 10 min, transferred in plastic tubes filled with a mixture of glycerol and 1 M sucrose (3:7), and immersed in liquid nitrogen within 1.5 min. The tubes were thawed in air at the room temperature, the embryos were washed in 0.5 M sucrose and cultivated in a Dulbecco medium with 20% fetal calf serum at 37 degrees C for 48 h or transplanted to recipient females. The survival of embryos was estimated according to their capacity to develop in vitro to the blastocyst stage or form normal fetuses after transplantation. After thawing and cultivation, 80.4% of NMRI embryos retained viability (80.7% in the control). For C57B1 and DBA embryos, these indices were 90.2 and 88.7%, and 59.3 and 66.7%, respectively. The differences between the experiment and control was insignificant for all strains. After thawing and transplantation of NMRI embryos, 34.4% developed into normal live fetuses (in the control 33.3%). At the same time, after cryoconservation by the standard method using a programmed freezer, the viability of ICR and NMRI embryos decreased approximately by 20% even in in vitro experiments. Thus, no differences were found in survival of the embryos of the studied strains after ultrafast freezing. This method proved to be very efficient for the embryos of both inbred and outbred mouse strains, since their capacity for development practically did not decrease after cryoconservation. The method may be used for production of cryobanks of embryos of various mouse strains.

[The preservation of mammalian ova and embryos at positive near-zero temperatures. II. The general patterns and mechanisms of hypothermic exposure]. / Khranenie iaitsekletok i émbrionov mlekopitaiushchikh pri polozhitel'nykh okolonulevykh temperaturakh. II. Obshchie zakonomernosti i mekhanizmy gipotermicheskogo vozdeistviia.

General patterns of the sensitivity of embryos to hypothermia are described, such as its species-specific dependence, its relationship with the embryonic stage, etc. Data are provided on differences in the cold resistance of the inner cell mass and trophectoderm cells, as well as that of the nucleus and cytoplasm. The mechanisms underlying the effects of cold on mammalian eggs and embryos are considered, and ways of improving the method of hypothermic storage are discussed. The general technological principles of the storage of mammalian embryos at positive near-zero temperatures are formulated for the first time. Advantages and drawbacks of the method and its possible practical application are discussed.

[The development of mouse embryos in vitro in a protein-free medium depending on the numbers of embryos in a microvolume of the medium]. / Razvitie myshinykh émbrionov in virto v srede bez belka v zavisimosti ot kolichestva zarodyshei v mikroob''eme sredy.

We studied the development of mouse embryos in vitro depending on the number of embryos in a given microvolume of the Ham's F-10 medium without protein or with the addition of serum. The absence of serum from the culture medium did not affect the development of two-cell embryos cultivate in groups of 5-6 (about 90% embryos developed until the stage of blastocyst and over 50% left zona pellucida), but the development of single embryos in the protein-free medium proceeded significantly worse. Single two-cell embryos cultivated in the serum-containing medium developed similar to embryos cultivated in groups. At the same time, no significant differences in development was found between eight-cell embryos cultivated individually or in groups of up to 10 embryos in the Ham's F-10 medium, either in the presence of serum or without it (about 95% embryos developed until the stage of blastocyst and over 70% left zona pellucida). The increase in the number of cultivated embryos over 10 had the adverse effect on development of either two-cell or eight-cell embryos. The attachment of blastocysts to the substrate after leaving zona pellucida and growth of trophectoderm was observed only in the presence of serum. These results suggest that interaction between preimplantation embryos in culture can probably be mediated by factor(s) released by embryos into the medium. Serum appears to contain such factor(s). Sensitivity of embryos to the factor(s) apparently depends on the stage of development.

[Role of ontogenetically different thymocytes in subpopulations of T-cells in releasing a humoral lymphocytic factor]. / Rol' ontogeneticheski razlichnykh timotsitov i subpopuliatsii t-kletok v vysvobozhdenii gumoral'nogo limfotsitarnogo faktora.

The activated mouse T-cells release in the culture medium a factor suppressing the ability of stem hemopoietic cells to form hemopoietic colonies in the spleens of lethally irradiated recipients. The factor of stem cell inhibition (FISC) obtained from the mouse thymocytes on the 1st, 7th and 10th day of postnatal life does not differ, by its inhibiting activity, from that obtained from the adult thymocytes, whereas the embryonic thymocytes under similar experimental conditions released FISC with a low inhibiting activity. A study of some physical-chemical and biological properties of FISC has shown that the latter is actively synthesized both by cortical and medullary thymocytes, has no (linear) specificity and resembles, by its characteristics, lymphokins of a wide effect, such as MIF and LT, rather than short-distant mediators, such as suppressor and helper factors.