General senescence of the adult organism is closely connected with reproductive one. Meanwhile, the age-related reduction in the female fertility is primarily associated with a decline in the gamete quality. Molecular and cellular changes in oocytes of old mammalian females are very similar to those occurring during aging of matured ova of their young counterparts, suggesting similarities in underlying mechanisms. The aim of the present work was to study actions of two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on age-associated modifications of metaphase-II (M-II) chromosomes in bovine oocytes using a model of the prolonged culture. We analyzed: (1) effects of PRL and GH on abnormal changes in the chromosome morphology in aging matured oocytes and the role of cumulus cells in these effects and (2) signaling pathways involved in the hormone actions. During the prolonged culture of oocytes, a gradual rise in the frequency of destructive modifications of M-II chromosomes was revealed. In the case of cumulus-enclosed oocytes (CEOs), PRL and GH exerted dose-dependent biphasic effects on the frequency of these modifications. Both PRL (50 ng/ml) and GH (10 ng/ml) decelerated the abnormal chromosome changes in CEOs, but did not affect the chromosome configuration in denuded oocytes. Concurrently, the presence of PRL and GH receptors in cumulus cells surrounding matured oocytes was demonstrated. Attenuating effects of both hormones on the chromosome modifications in aging CEOs were abolished by PP2 (an inhibitor of Src-family tyrosine kinases), triciribine (an inhibitor of Akt kinase), and calphostin C (a protein kinase C inhibitor). Our findings indicate that PRL and GH can exert the similar decelerating action on age-associated alterations in the M-II chromosome morphology in bovine ova, which is mediated by cumulus cells and may be related to activation of Src-family tyrosine kinases as well as Akt- and protein kinase C-dependent signal pathways.

Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway.

Centrosome formation during early development in mice and rats occurs due to the appearance of centrioles de novo. In contrast, in humans and other non-rodent mammals, centrioles are thought to be derived from spermatozoa. Ultrastructural study of zygotes and early embryos of cattle at full series of ultrathin sections show that the proximal centriole of the spermatozoon disappears by the end of the first cleavage division. Centrioles appear in two to four cell embryos in fertilized oocytes and in parthenogenetic embryos. Centriole formation includes the appearance of atypical centrioles with randomly arranged triplets and centrioles with microtubule triplets of various lengths. After the third cleavage, four centriolar cylinders appear for the first time in the blastomeres while each embryo still has two atypical centrioles. Our results showed that the mechanisms of centriole formation in different groups of mammals are universal, differing only in the stage of development in which they occur.

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Centrosome formation during early development in mice and rats occurs due to the appearance of centrioles de novo. In contrast, in humans and other non-rodent mammals, centrioles are thought to be derived from spermatozoa. Ultrastructural study of zygotes and early embryos of cattle at full series of ultrathin sections showed that the proximal centriole of the spermatozoon disappears by the end of the first cleavage division. Centrioles appear in 2-4 cell embryos in fertilized oocytes and in parthenogenetic embryos. Centriole formation includes the appearance of atypical centrioles with randomly arranged triplets and centrioles with microtubule triplets of various lengths. After the third cleavage, four centriolar cylinders for the first time appear in the blastomeres while each embryo still has two atypical centrioles. Our results showed that the mechanisms of centriole formation in different groups of mammals are universal, differing only in the stage of development in which they occur.

Serratia plymuthica strain IC1270 isolated from the rhizosphere, possessing antagonistic activity towards a wide range of plant-pathogenic fungi, is able to hydrolyze phytate. Phytase activity was found intracellularly, while no activity was detected in the culture liquid. Optimum activity was found at pH 4-5; it completely disappeared at pH > 7.0 and 2.5. Phytase production was practically absent in the exponential phase and reached a maximum in the late stationary phase. Mutations of genes grrA and grrS, encoding GacA/GacS-like 2-component global regulatory system, or in gene rpoS encoding the sigma factor RpoS subunit of RNA polymerase, led to a deficiency in phytase production. Introduction into mutants of the respective wild-type genes cloned into the wide-range plasmid pJFF224-NX under the control of the bacteriophage T4 gene 32 promoter complemented this deficiency. This is the first report implicating the GacA/GacS global regulators and RpoS factor in phytase production in bacteria.

A method for the in vitro evaluation of the toxic effects occurring in human cell lines upon the expression of genes from a range of pathogens is proposed. The method is based on the transient expression of the genes in the HEK293 cell line. Induction of cell death upon the expression of the gene coding for pro tease 3C from the human hepatitis A virus has been demonstrated for the first time using the method pro posed. Expression of the gene coding for protease 2A from human poliovirus has also been shown to induce cell death, while cathepsins B and D did not have a cytotoxic effect on the culture used.

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