Production of a Cloned Offspring and CRISPR/Cas9 Genome Editing of Embryonic Fibroblasts in Cattle.

Somatic Cell Nuclear Transfer (SCNT) technique was used to produce the first viable cloned cattle offspring in Russia. Whole-genome SNP genotyping confirmed that the cloned calf was identical to the fibroblast cell line that was used for SCNT. CRISPR/Cas9 approach was subsequently used to knock out genes for beta-lactoglobulin gene (PAEP) and the beta-lactoglobulin-like protein gene (LOC100848610) in the fibroblast cells. Gene editing (GE) efficiency was 4.4% for each of these genes. We successfully obtained single-cell-derived fibroblast colonies containing PAEP and LOC100848610 knockouts, which will be used to produce beta-lactoglobulin-deficient cattle.

[Dynamics of morphofunctional changes in aging bovine ova during the prolonged culture in vitro].

In the absence of activating stimuli, aging processes are initiated in matured mammalian oocytes, which negatively affect the quality of ova and their capacity for further development. On the model of the prolonged culture of bovine oocytes, the dynamics of a number of morphofunctional changes associated with the postovulatory aging was investigated in the present work. In cumulus-enclosed oocytes, migration of the first polar body relative to metaphase II chromosomes started between 18 and 22 h of maturation. The angle of the body deviation from the metaphase plate rose as the culture time increased to 30 h. By 32 h of culture, a gain in the rate of ova with the abnormal chromosome morphology was observed that continued up to 56 h. Furthermore, after 56 h, signs of spontaneous parthenogenetic activation were revealed in 16 percent of matured oocytes. During the prolonged culture of oocytes deprived of cumulus cells after 20 h of maturation, an increase in the frequency of chromosomal abnormalities was found only by 44 h. At the same time the cumulus elimination did not affect the maintenance of the meiosis II blockade in aging ova. Meanwhile, destructive chromosomal changes in oocytes were attended by a gradual rise in the level of apoptotic degeneration and reduction in the proliferative activity of surrounding cumulus cells. The results obtained point to the various temporal dynamics of distinct morphofunctional changes and to the participation of cumulus cells in modulation of the speed of metaphase chromosome destructive modifications in aging bovine ova.

[Role of protein kinases in prolactin signaling in bovine oocyte-cumulus complexes].

Mechanisms of prolactin signal transduction in generative and somatic cells of mammalian ovarian follicles have been studied only to a small extent. In the present work, the involvement oftyrosine kinases and protein kinase C in mediating of the previously revealed modulating effects of prolactin on the nuclear maturation of bovine oocytes and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. Tyrosine kinase inhibitor, genistein, was found to suppress the stimulating action of prolactin on the completion of oocyte nuclear maturation and cumulus expansion, whereas protein kinase C inhibitor, calpostin C, did not affect these hormonal effects. Furthermore, both genistein and calpostin C inhibited the inducing influence of prolactin on the proliferative activity of cumulus cells. At the same time the retarding action ofprolactin on destructive processes in cumulus cells was blocked only in the presence of calpostin C. The results of the study suggest that the stimulatory influence of prolactin on oocyte nuclear maturation and attendant cumulus expansion is achieved with the participation of tyrosine kinases, whereas the modulating action of the hormone on the functional state of cumulus cells depends on activation of not only tyrosine kinases, but also protein kinase C.

[Realization pathways of prolactin modulating influence on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes].

Cyclic AMP is one of the key regulators of mammalian meiosis. In the present work, realization pathways of the previously revealed modulating influence of prolactin (PRL) on the cAMP-dependent mechanisms of meiosis regulation in bovine oocytes were studied. A comparative investigation of individual and combined effects of PRL (50 ng/ml) and an activator of adenylate cyclase forskolin (FK. 20 microM) on the meiotic reinitiation and nuclear maturation completion in cumulus-enclosed and cumulus-free oocytes was performed. It has been shown that the pattern of PRL influence on the meiotic resumption in oocytes devoid of cumulus cells depends on the presence of FK in the culture medium. Furthermore, realization of this influence is not associated with the activation of cytoplasmic isoforms of protein kinase C. It has been also found that PRL inhibits the retarding action of FK on the completion of oocyte nuclear maturation in both the presence and absence of cumulus cells. The findings suggest that PRL may modulate the functioning of the cAMP-dependent mechanism of meiosis regulation by the direct action on bovine oocytes, with realization of the action being independent of the metabolic coupling of oocytes with cumulus cells.

[Interaction of signal cascades induced by cAMP and prolactin in bovine oocyte-cumulus complexes].

Prolactin (PRL) is one of the pituitary hormones participating in the control of mammalian folliculo- and oogenesis. In the present study, the joint effect of PRL (50 ng/ml) and dibutyryl cAMP (dbcAMP, 1 mM) on oocyte maturation and the morphologic-functional state of surrounding cumulus cells was investigated in vitro. It has been shown that PRL totally suppresses the braking impact of dbcAMP on meiosis reinitiation and the completion of oocyte nuclear maturation. Furthermore, PRL partly inhibited cumulus expansion induced by dbcAMP, although it exerted the opposite effect in the control medium. In the presence of PRL, the inhibitory impact of dbcAMP on the proliferative activity of cumulus cells and on the PRL-elicited braking of destructive processes in the cells has been found. In cumulus cells, mRNA expression of PRL receptor long isoform was revealed by the RT-PCR method. The data obtained suggest an interaction of signal cascades induced by PRL and cAMP in bovine oocyte-cumulus complexes, with the coupling site of these cascades in oocytes being apparently different from that in cumulus cells.