[Evaluation of Indicators of Postejaculation Maturation of Spermatozoa of Bos taurus Using a Chlortetracycline Test].

Postejaculation maturation of spermatozoa (capacitation and acrosomal exocytosis) in bovine sperm was assessed using the method of staining with chlortetracycline and inhibitor analysis to identify the transition ways of calcium of intracellular depots under the influence of highly dispersed silica. It was shown that highly dispersed silica in a concentration of 0.001% stimulates capacitation but has no impact on the acrosome exocytosis in bovine sperm. Activated by highly dispersed silica, capacitation was inhibited in the presence of cytochalasin D and H-89 inhibitors, whereas nocodazole and Ro 31-8220 had no influence on this process. The joint action of theophylline and guanosine diphosphate stimulates an increase in the amount of capacitated sperm cells, similarly to highly dispersed silica; inhibitors cytochalasin D and H-89 restrict the capacitation of sperm activated by these compounds. At the same time, the joint effect of prolactin and guanosine triphosphate had no effect on the capacitation of spermatozoa; addition of nocodazole and Ro 31-8220 inhibitors did not alter the effect of prolactin and guanosine triphosphate on the capacitation of sperm. A hypothesis was put forward, according to which increase in the cryoresistance of spermatozoa of bulls under the influence of highly dispersed silica is, apparently, determined by the transition of Ca2+ between the intracellular stores in the direction from inositol triphosphate-sensitive to inositol triphosphate-insensitive intracellular stores of calcium. The obtained data allow us to expand the notion of the biochemical mechanisms of capacitation in the spermatozoa of bulls.

[Effect of vitrification on the functioning of the structural elements of the cytoskeleton in porcine oocytes].

On the basis of inhibition analysis using inhibitors of protein kinase A and polymerization of microfilament under the influence oftheophylline, GDP and GTP have studied the release of Ca2+ from intracellular stores (ryanodin- and inositol-1 ,4,5-trisphosphate) of native and devitrified porcine oocytes. It is found that theophylline, as well as GDP stimulate the release of Ca2+ from intracellular stores in the native and devitrified oocytes, but a further release of Ca2+ from intracellular stores under the joint action of theophylline and GDP was observed only in native oocytes. Inhibitors of protein kinase A and polymerization of microfilaments in native oocytes have a negative impact on the further release of Ca2+ from intracellular stores under the joint action of theophylline and GDP. In possible transition of Ca2+ between intracellular depots, that stimulated by GDF, but GTF doesn't take part. The received results indicate that disruption of the functioning of the system of microfilaments in oocytes determined by vitrification that assumes a priori difference in the signal transduction pathways in native and devitrified oocytes at diplotene stage.

[Identification of mechanisms of the calcium signaling at influence of estradiol on porcine oocytes, stimulated by theophillin and somatotropin].

Through the use of inhibitory analysis by using the fluorescent probe chlortetracycline examined the effects of estradiol on the mobilization of Ca2+ from intracellular stores of porcine oocytes stimulated by growth hormone and theophylline. It is shown that somatotropin or theophylline stimulates the mobilization of Ca2+ from intracellular stores of oocytes in control group, while their combined action does not lead to an additional exit of Ca2+ from intracellular stores. Inhibitor of protein kinase C had no effect on the release of Ca2+--from oocyte stimulated by growth hormone or theophylline. In estradiol-treated oocytes, only the combined effects of growth hormone and theophylline stimulates the release of Ca2+ from intracellular stores, and that the release of Ca2+ is reduced by the use of an inhibitor of protein kinase C. In the presence of estradiol, an inhibitor of microtubule polymerization blocked the release of Ca2+ stimulated by the combined action of growth hormone and theophylline. Incubation of oocytes in the medium with the subsequent addition of ADP and GDP have an inhibitory effect on the release of Ca2+ from intracellular stores, stimulated joint action of growth hormone and theophylline. The findings suggest the involvement of estradiol in the mechanisms of calcium signaling in porcine oocytes.

[Ca2+ exit from intracellular stores of growing and fully grown native and devitrified porcine oocytes].

The exit of Ca2+ from intracellular stores in growing and fully grown native and devitrified porcine oocytes stimulated by somatotropin and GTP was investigated using fluorescent dye chlortetracycline. In native as well as in the devitrified porcines oocytes, in their fully grown phase, joint action of somatotropin and GTP stimulated additional freeing of Ca2+ from intracellular stores, but during subsequent processing of the cell with nocodazole (a polymerization inhibitor of microtubules), additional exit of calcium did not occur. In the growing phase of native oocytes during the joint acting of somatotropin and GTP additional exit for Ca2+ from the intracellular stores did not occur. Join action of somatotropin and GTP on growing devitrified oocytes lead to the additional freeing of Ca2+ from intracellular stores. Injection of nocodazole inhibited the exit for calcium in growing devitrified oocytes treated with somatotropin and GTP. The data obtained points to the absence of difference of signal transduction mechanisms in growing and fully grown oocytes after devitrification.

[Identification of signal transduction pathway in fresh and vitrified porcine oocytes].

Signal transduction pathway under the influence of somatotropin have been identified basis on the analysis of Ca2+ release from intracellular stores of fresh and vitrified porcine oocytes using inhibitory analysis. Somatotropin and GTP individually stimulated Ca2+ release from intracellular stores. The joint action of somatotropin and GTP activated additional Ca2+ release from intracellular stores both in fresh and vitrified porcine oocytes. Treatment of the oocytes with inhibitor of protein kinase C caused no additional Ca2+ release from intracellular stores. Ca2+ release from intracellular stores stimulated by GTP was connected with phosphate hydrolysis. Moving between intracellular Ca2+ depots stimulated by GTP was not determined by phosphate hydrolysis. Inhibitor of protein kinase C and microtubules were involved in the interaction of various intracellular depots. The data obtained suggest that signal transduction pathway in porcine oocytes do not change after vitrification.

[Ca2+ release from intracellular stores of pig oocytes during different stages of growth].

Ca2+ release from intracellular stores of pig oocytes was investigated using the Ca(2+)-sensitive fluorescent dye chlorotetracycline. Oocytes were divided into growing ones and those that completed their growth using brilliant cresyl clue (BCB) staining. The stained oocytes (BCB "+") were determined as the ones that completed their growth, while the stainless ones (BCB "-") were determined as those in the final stages of growth. In the BCB "+" and BCB "-" oocytes, prolactin, theophylline, GTP, and GDP cause Ca2+ to exit intracellular stores. In the oocytes that completed their growth, joint action of prolactin and GTP activates additional release of Ca2+, in which protein kinase C takes part. In growing oocytes, joint action of prolactin and GTP does not lead to additional release of Ca2+. Joint action of theophylline and GDP in growing oocytes and oocytes that completed the growth stage promotes additional Ca2+ exit from intracellular stores. This exit is regulated by protein kinase A. The obtained data show that there various routes of Ca2+ release from intracellular stores in growing and grown pig oocytes.

[Effect of testosterone in Ca2+ mobilization in oocytes and porcine granulose stimulated by prolactin and theophylline].

Influence of testosterone in Ca2+ exchange in oocytes and porcine granulose stimulated by prolactin and theophylline was investigated using fluorescent dye chlortetracycline. It was shown that prolactin and theophylline separately have not affected on the Ca2+ exit from intracellular stores of oocytes. Prolactin and theophylline in common stimulate exit of Ca2+ from intracellular stores in oocytes, which decreased at presence of inhibitor of protein kinase A. Ca2+ exit from intracellular stores of oocytes stimulated by prolactin and theophylline in common was inhibited by thapsigargin. In the presence of testosterone prolactin and theophylline separately stimulate increasing of cytoplasmic Ca2+ concentration and exit of Ca2+ from intracellular stores of granulose. Prolactin and theophylline in common activate additional exit of Ca2+ from intracellular stores in granulose, which decreased in presence of inhibitor of protein kinas C. The date obtained suggest differences in regulated mechanisms of cell signaling in porcine granulose and oocytes.

[Effect of estradiol on fluctuation of calcium in swine granulose cells stimulated by theophylline and prolactin].

Effect of estradiol on Ca2+ exit from intracellular stores in swine granulose cells activated by theophylline and prolactin is investigated with using fluorescent Ca2+ sensitive probe chlortetracycline. It was shown that prolactin and theophylline stimulated Ca2+ exit from intracellular stores. In absence of estradiol if we treatment of oocytes by theophylline and prolactin together we have not found influence at Ca2+ exit from intracellular stores. In the presence of estradiol we had opposite results: we tested additional Ca2+ exit at common action of theophylline and prolactin. Protein kinase C influences on Ca2+ exit from intracellular stores during common affect of theophylline and prolactin only in the presence of estradiol. Protein kinase A does not influence this process. The obtained data indicate an influence of estradiol on Ca2+ exit from intracellular stores in swine granulose cells stimulated by joint action of prolactin and theophylline.

[Antigenicity and B-epitope mapping of hepatitis C virus envelope protein E2].

Immunogenicity for laboratory animals (rabbits and mice) of the whole hepatitis C virus envelope proteins and their conserved as well as hypervariable HVR1 sites has been investigated. Rabbit immune responses to HCV envelope proteins (both single E2 and E1E2 heterodimer) were shown to be much more efficient than murine immune responses. Upon the immunization of the rabbit with E2 protein, antibodies to several highly conserved linear B-epitopes of this protein as well as to the N-terminal fragment of the hypervariable region HVRI were formed. Epitopes in the CR2 region were determined for the first time. Cross-reactivity was revealed between the N-terminal fragment of the protein E2 hypervariable region HVRI and the octapeptide fragment of the protein E1 conserved region CR1, which shared four identical amino acid residues.

[Effect of estradiol on Ca2+ release from intracellular stores in porcine oocytes stimulated by prolactin, theophylline, or guanosine triphosphate].

The interaction between prolactin and theophylline as well as between prolactin and guanosine triphosphate during Ca2+ release from intracellular stores of estradiol-treated porcine oocytes isolated from the ovary at the stage of follicular growth were studied using fluorescent Ca(2+)-sensitive probe chlortetracycline. In the absence of estradiol, prolactin or theophylline induced Ca2+ release from intracellular stores; however, no increase in Ca2+ release was observed after their combined action. Conversely, Ca2+ release from intracellular stores increased only after the combined exposure to prolactin and theophylline in the the presence of estradiol. In the absence of estradiol, guanosine triphosphate induced calcium release alone and together with prolactin. Protein kinase C regulated Ca2+ release from intracellular stores after the combined exposure to prolactin and theophylline only in the presence of estradiol; while the activation of protein kinase C required no estradiol during the combined exposure to prolactin and guanosine triphosphate. The data obtained indicate the effect of estradiol on Ca2+ release from intracellular stores after the combined exposure to prolactin and theophylline, while no such effect was observed after the combined exposure to prolactin and guanosine triphosphate.

[Influence of ryanodine and inositol trisphosphate receptors inhibitions on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP].

The influence of ryanodine and inositol triphosphate receptors inhibitors on Ca2+ exit from intracellular stores of porcine oocytes stimulated by prolactin and GTP was investigated using fluorescent dye chlortetracycline. Porcine oocytes were isolated from ovaries with yellow body. Ca2+ exit from intracellular stores of porcine oocytes activated by prolactin (5 and 50 ng/ml) in calcium free medium was decreased after treatment of oocytes by heparin (inhibitor of inositol triphosphate receptors) and was not changed after treatment of oocytes by ruthenium red (inhibitor of ryanodine receptors). Inhibition of protein kinase C did not affect on the Ca2+ exit stimulated by prolactin. GTP did not stimulate Ca2+ exit from intracellular stores of pig oocytes, and inhibitors of both calcium channels and proteinkinase C had no influence on this process. The joint action of prolactin and GTP did not result in additional Ca2+ exit from intracellular stores of oocytes after both pretreatment and untreatment by the inhibitor of protein kinase C. The data obtained testify to activation of IP3-sensitive receptors under effect of prolactin and in the absence of GTP influence on these receptors.

[Prolactin modulation of theophylline inhibitory action on maturation of bovine oocyte-cumulus complexes in vitro].

The comparative investigation of the individual and joint impact of prolactin (PRL, 50 ng/ml) and theophylline (TP), a nonselective inhibitor of phosphodiesterases, on nuclear maturation of bovine oocytes and the expansion of cumulus cells enclosing the oocytes was carried out using a model of in vitro culturing. It has been shown that TP (5 mM) exerts a short-term inhibitory action on oocyte meiosis reinitiation and blocks it at diakinesis and metaphase I stages as well as inhibits the cumulus expansion. The addition of PRL to the medium containing TP caused the decrease in the rate of oocytes at diplotene stage after 6 h of culturing and the increase in the rate of oocytes attained the closing stages of maturation after 24 h of culturing. Furthermore, PRL suppressed partly the inhibitory impact of TP on the expansion of cumulus cells. The data obtained suggest the signal cascade induced by PRL in bovine oocyte-cumulus complexes to be compled with cAMP-dependent intracellular pathway.

[Effects of guanine nucleotides and protein kinase C on prolactin-stimulated release of Ca from intracellular stores of pig oocytes].

The effects of guanine nucleotides and protein kinase C on prolactin-stimulated Ca2+ release from intracellular stores of pig oocytes were studied using the fluorescent dye chlorotetracycline. The effect of prolactin was related to the protein kinase C activation. Inhibition of protein kinase C stimulated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin in the presence of extracellular Ca2+ and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. In a Ca2+-free medium, prolactin did not stimulate Ca2+ release from intracellular stores of the oocytes treated with GDP in the presence of GDP. GTP inhibition of protein kinase C activated Ca2+ release from intracellular stores of the pig oocytes treated with 5 ng/ml prolactin and inhibited Ca2+ release from intracellular stores of the pig oocytes treated with 50 ng/ml prolactin. These data suggest the influence of guanine nucleotides and protein kinase C on calcium metabolism, stimulated by prolactin.

[Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes].

Effect of estradiol on stimulated theophylline and prolactin Ca2+ exit from intracellular stores of pig oocytes was investigated using fluorescent dye chlortetracycline. It was shown that in the presence of estradiol neithert theophylline nor prolactin stimulated Ca2+ exit from intracellular stores of oocytes. Unlike, the common action oftheophylline and prolactin, also in the presence of estradiol, stimulated Ca2+ exit from intracellular stores. Inhibition of protein kinase C inhibits Ca2+ exit from intracellular stores in common action of theophylline and prolactin. These data suggest an obvious influence of estradiol on Ca2+ exit from intracellular stores of pig oocytes stimulated by theophylline and prolactin.

[Dependence of Ca2+ release from intracellular stores on NADH and FAD levels in fertilized and unfertilized bovine oocytes].

Relation between NADH and FAD concentrations and the quantity of calcium released from intracellular stores in fertilized and unfertilized bovine oocytes was investigated using luminescent analysis. Inhibition of Ca2+ exit from intracellular stores was detected in degenerative oocytes at metaphase II and 2-cell embryos. The intensity of both NADH and FAD fluorescence increased in 2-cell degenerated embryos, whereas the increase in only NADH fluorescence intensity occurred in degenerated oocytes at metaphase II stage. Degeneration exerted no influence on NADH fluorescence intensity or Ca2+ exit from intracellular stores, whereas a decreased FAD fluorescence intensity was noted in degenerated pronuclei. The obtained data testify that in degenerated zygotes and early embryos Ca2+ release may occur from different intracellular stores.

[Peculiarities of theophylline and prolactin interaction and calcium fluctuation from intracellular stores of porcine oocytes in the presence of estradiol].

Using a fluorescent dye chlortetracycline, a study was made of the effect of estradiol on the interaction of theophylline and prolactin in the course of Ca2+ exit from intracellular stores of pig oocytes, isolated from ovaries at the stage of follicle growth. It is shown that in the presence of estradiol, prolactin does not stimulate Ca2+ exit from intracellular stores of pig oocytes. The action of theophylline similarly does not stimulate Ca2+ exit. Unlike, a joint effect of theophylline and prolactin on pig oocytes in the presence estradiol stimulated Ca2+ exit from intracellular stores of pig oocytes. These data demonstrated the influence of estradiol on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.

[Participation of granulosa cells in mediation of prolactin and somatotropin action on bovine oocyte-cumulus complexes in vitro].

Maturation of bovine oocyte-cumulus complexes (OCC) in media derived following granulosa cell culturing with prolactin (PRL, 50 ng/ml) and somatotropin (ST, 10 ng/ml) was studied. A medium conditioned by granulosa cells in the presence of PRL or ST exerted a stimulating effect on the proliferative activity of cumulus cells. ST introduction into the granulosa cell culture also caused a decrease in the rate of cumulus cells with degenerated chromatin at a subsequent OCC culturing. At the same time, the expansion of cumulus did not depend on hormone availability in the culture medium for granulosa cells. When OCC matured in conditioned media, a short-term inhibition of oocyte meiosis reinitiation (after 6 h of culturing) was revealed in both the experimental groups, as compared to the control. Furthermore, the addition of ST and PRL to granulosa cell culture resulted in a subsequent decline in the rate of oocytes with signs of chromosome degeneration, observed as early as by 6 h of incubation and to be retained throughout the whole period of OCC culturing. In this case the earlier resumption of meiosis was associated with a higher rate of degeneration of the nuclear material in oocytes. The results of the present study suggest that granulosa cells may mediate, at least in part, PRL and ST impacts on in vitro maturation of bovine OCC, with no contact between OCC and granulosa cells being required for hormonal signaling.

[Prolactin and somatotropin binding by granulosa cells of cows at different reproductive states]. / Sviazyvanie prolaktina i somatotropina kletkami granulezy korov pri ikh raznom reproduktivnom sostoianii.

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3-5 to 6-10 mm, specific binding of 125I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data suggest that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1-2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.

[Prolactin-binding activity of bovine granulosa cells on luteinization and exposure to somatotropin in vitro]. / Prolaktinsviazyvaiushchaia aktivtost' kletok granulezy korov pri liuteinizatsii i vozdeistvii somatotropina in vitro.

The influence of luteinization and bovine somatotropin (ST, 5-50 ng/ml) during cultivation of bovine granulosa cells on their ability to bind [125I]-labeled bovine prolactin (PRL) was studied. On the second day of cultivation in serumfree medium, granulosa cells from immature antral follicles underwent spontaneous luteinization, in both the absence and presence of ST. The level of [125I]-PRL specific binding to cells increased after two days of cultivation, with a negative correlation being revealed between estradiol production by the cells and their PRL-binding activity. At the same time, the addition of ST to the culture medium had no effect on the level of [125I]-PRL specific binding to native and luteinizing granulosa cells. The findings suggest a stimulatory influence of the luteal differentiation process on the PRL-binding activity of bovine granulosa cells, this influence is independent of the action of ST.