[Hormone-Induced In Vitro Maturation and Ovulation of Danio rerio Oocytes and Production of Eggs Capable of Fertilization and Futher Development].

It is common knowledge that zebrafish, Danio rerio, oocytes in their follicular envelope that have reached definitive size undergo in vitro maturation in 90% Leibovitz's medium, pH 9.0, when treated with 17α,20ß-dihydroxyprogesterone and acquire developmental competence but do not ovulate (Seki et al., 2008). We have demonstrated that zebrafish oocytes that have undergone maturation under the indicated conditions ovulate when treated with prostaglandin F2α (5 µg/mL) and/or 20% carp ovarial fluid and are capable of development towards the actively feeding larvae upon fertilization (the maximum follow-up period).

[Hormonal Induction of In Vitro Maturation and Ovulation of Loach Oocytes and Obtaining Egg Cells Capable of Fertilization and Development].

Loach oocytes that have reached a definitive size and are surrounded by follicular envelopes are capable of maturation and ovulating under the effect of 1 µg/mL progesterone in 75% Leibovitz medium with 1 g/L sodium bicarbonate or with pH adjustment to 9.0 by 1 N sodium hydroxide. Inseminated eggs are developed until the stage of when adding 20% bovine serum to the incubation medium. Substitution of the bovine serum with 10-20% loach ovarian fluid or 20% carp ovarian fluid provides more complete development of inseminated eggs until the stage of prolarvae that pass to active nutrition.

[Nonhormonal stimulation in vitro of oocyte maturation in sturgeons].

Incubation of sturgeon full-grown ovarian follicles in amphibian Ringer solution with increased sodium bicarbonate concentration results in "spontaneous" oocyte maturation. Addition of sodium bicarbonate to diluted Leibovitz medium also induces maturation of follicle-enclosed oocytes. Effective threshold concentration of sodium bicarbonate depends on the composition of culture medium and, especially, on the physiological state of follicle-enclosed oocytes. As evidenced by experiments with actinomycin D, oocyte maturation induced by bicarbonate ions does not depend on RNA synthesis. An attempt was made to elucidate the involvement of steroidogenesis in bicarbonate ion-induced oocyte maturation. Surprisingly, the inhibitors used, such as aminogluthetimide, diltiazem, andestradiol-17ß, not only did not inhibit but also enhanced oocyte maturation. Manual removal of follicle envelopes demonstrated that denuded oocytes retained the ability to mature in a culture medium with increased sodium bicarbonate concentration. However, the range of effective bicarbonate ion concentrations for denuded oocytes is more restricted than for the follicle-enclosed oocytes. A hypothesis of competition of different processes occurring in the ovarian follicle for energy resources is proposed to explain the revealed paradoxical effect of substances affecting steroidogenesis.

[Role of hydration in ovulation of common frog oocytes in vitro].

Stimulation of ovulation of the common frog Rana temporaria oocytes with homologous pituitary extract caused an increase in their volume. Factors that are known to inhibit hydration in teleostean oocytes (potassium-free Ringer solution and inhibitor of Na+, K(+)-ATPase--ouabain), as well as use of aquaporin inhibitors (mercuric chloride and methylmethanethiosulphonate) inhibited also homologous pituitary extract-induced volume increase in follicle-enclosed oocytes and let to reduced percentage of ovulated oocytes. Volume of denuded oocytes remained unchanged in the course of maturation when exposed to progesterone or other treatments. The data obtained suggest that stimulation ofoocyte ovulation in the common frog caused an increase in their hydration that in necessary for their ovulation but this did not occur in denuded cells.

[Stimulation in vitro of oocytes of acipenserids with progesterone and homologous gonadotropic hormone of hypophysis].

We showed that the percentage of oocytes of acipenserids ovulating in vitro in Ringer solution modified for sturgeons (RMS) considerably depends on the concentration of sodium bicarbonate and the concentration of progesterone. Under optimal conditions (0.5 g/L of sodium bicarbonate and 30 ng/mL of progesterone), it can be higher than 80. Oocytes that matured and ovulated under such conditions are capable of normal development. In the best case, approximately 70% of developing embryos (of the number of ovulated oocytes) reach the stage of hatching (dead-line of observation). This method of producing offspring based on the insemination of oocytes that have matured and ovulated in vitro can be used in work with single females of rare and disappearing species of acipenserids.

[Hydration of oocytes in bony fishes].

Data are collected on the hydration of oocytes in bony fishes during maturation stimulated by gonadotropic or steroid hormones in vivo and in vitro. The reasons for hydration, its dynamics, and certain of the mechanisms ensuring income of water and ions into the oocyte are considered.

[In vitro stimulation of oocyte ovulation in teleosts by gonadotropic and steroid hormones].

Published data on in vitro stimulation of oocyte maturation and ovulation by gonadotropic and steroid hormones in different teleost species are reviewed. The involvement of meiosis-inducing steroids, eicosanoids, and nuclear progesterone receptor in the mechanism of ovulation induction is considered.

[Aquaporins in gametogenesis of vertebrate animals].

A review of the data on the presence, localization, and supposed role of aquaporin water channels in oocytes of Xenopus laevis, oogenesis and maturation of teleosts Sparus auratus and Oncorhynchus mykiss, oogenesis and oocyte maturation of rats and mice, and spermatogenesis of several mammalians.

[Influence of culture medium osmolality on maturation and ovulation of Rana temporaria oocytes stimulated in vitro by the pituitary gland extract or progesterone].

The influence of diluted Ringer solution on ovulation and maturation of common frog oocytes stimulated in vitro by homologous pituitary extract (0.005 pit/ml) or progesterone (1 pg/ml) was studied. During wintering, the dilution of Ringer solution led to a decreased percentage of oocytes ovulated and matured under the influence of both inducers. As the season of reproduction approached, the dependence of oocyte maturation and ovulation on the Ringer solution dilution weakened. Possible causes of different dependence of the ovulation of amphibian and sturgeon oocytes stimulated by gonadotropic hormones or progesterone on the culture medium osmolality is discussed.

[Involvement of gap junctions in stimulation of in vitro maturation of the common frog oocytes by low progesterone concentrations]. / Uchastie shchelevykh kontaktov v stimuliatsii sozrevaniia ootsitov travianoi liagushki in vitro nizkimi kontsentratsiiami progesterona.

The inhibitor of gap junctions 18alpha-glycerretinic acid inhibits the maturation of follicle-enclosed common frog oocytes stimulated by low progesterone concentrations. The inhibitory effect of 18alpha-glycerretinic acid does not depend on concentrations within the limits of 5-40 microM. The inhibitory progesterone concentrations differ markedly in different females. The inhibitory effects of actinomycin D (5 microg/ml) and 18alpha-glycerretinic acid (5 microg/ml) were expressed when the same progesterone concentrations were used. When injected in an intact oocyte, Lucifer yellow was transported into the follicle cells, thus suggesting the presence of gap junction between these latter and the oocyte. The data obtained suggest that the previously described transcription-dependent factor formed in the follicle cells under the influence of low progesterone concentrations and stimulating oocyte maturation is transported in the oocyte along gap junctions.

[Maturation of the follicle-enclosed common frog oocytes stimulated by low progesterone concentrations depends on transcription]. / Sozrevanie okruzhennykh follikuliarnymi obolochkami ootsitov travianoi liagushki, stimulirovannoe nizkimi kontsentratsiiami progesterona, zavisit ot transkriptsii.

The maturation of follicle-enclosed common frog oocytes stimulated by low progesterone concentration is inhibited by actinomycin D (5 micrograms/ml). The concentrations of progesterone, at which oocyte maturation was inhibited by actinomycin D, varied with the season and were different in different females. The inhibitor of steroidogenesis aminogluthetimide (100 micrograms/ml) did not suppress the maturation of follicle-enclosed oocytes in most experiments, which was induced by both high and low progesterone concentrations. The induction of maturation of the defolliculated ("denuded") oocytes required lower progesterone concentrations than of the follicle-enclosed oocytes and, in addition, the maturation of denuded oocytes was not suppressed by actinomycin D. Thus, it was shown for the first time that low doses of progesterone induced the maturation of amphibian oocytes while acting on the follicle wall cells and this process depended on transcription. The factor inducing or enhancing maturation, which is formed in the follicle cells in the presence of low progesterone concentrations, remains as yet unknown.

[Effects of the antagonist of the classical progesterone receptor on ovulation and maturation of common frog (Rana temporaria L.) oocytes in vitro]. / Vliianie antagonista klassicheskogo retseptora progesterona mlekopitaiushchikh na ovuliatsiiu i sozrevanie ootsitov travianoi liagushki in vitro.

Progesterone induces maturation and ovulation of amphibian oocytes and, in the process, binds to the receptors in oocytes and follicle cells. It is still unclear to what extent the progesterone receptors in these cells are similar. We tried to answer this question using RU486, an antagonist of the classical mammalian progesterone receptor. Pretreatment of the ovarian follicles with RU486 (1-20 microM) does not affect the maturation or ovulation of the common frog oocytes stimulated in vitro by pituitary hormones or progesterone and even increases (at low concentrations) the percentage of maturated and ovulated oocytes. In addition, RU486 (5-40 microM) stimulates the oocyte maturation and ovulation in a dose-dependent manner. Pretreatment of the follicles with actinomycin D (5 microM) inhibits both RU486-stimulated processes. An increase in RU486 concentration overcomes the inhibitory effect of actinomycin D on oocyte maturation. On the contrary, the inhibitory AD effect on oocyte ovulation does not depend on the RU486 concentration. RU486 stimulates the maturation of both follicle-enclosed and "denuded" oocytes. However, the sensitivity of the latter to RU486 is much lower and its effect on the denuded oocyte is not inhibited by actinomycin D. The data obtained suggest that in amphibians, unlike in mammals, RU486 is an agonist of progesterone. It stimulates the transcription-independent maturation of denuded oocytes (membrane receptor) and transcription-dependent ovulation (classical receptor). In addition RU486 can stimulate (at certain doses) the transcription-dependent maturation of follicle-enclosed oocytes.

[The role of chloride channels and chloride ions in steroidogenesis regulation in the gonads of amphibians, birds and mammals]. / Rol' khlornykh kanalov i ionov khlora v reguliatsii steroidogeneza v gonadakh amfibii, ptits i mlekopitaiushchikh.

The paper represents the first review of data on the involvement of chloride channels (their inhibitors and media, in which chloride ions are substituted for anions that poorly penetrate in the cell) in the regulation of basal and gonadotropin-stimulated steroidogenesis in the gonads of amphibians, birds, and mammals. Possible causes are considered for different reactions of the gonad steroidogenic cells in representatives of different vertebrate classes to a decreased medium concentration of chloride and the involvement of chloride channels and/or chloride ions in the regulation of steroidogenesis is discussed.

[Role of protein kinase A and calcium ions in regulating ovulation of oocytes by gonadotropins in the grass frog (Rana temporaria) in vitro]. / Rol' proteinkinazy A i ionov kal'tsiia v reguliatsii gonadotropinami ovuliatsii ootsitov travianoi liagushkin (Rana temporaria) in vitro.

The inhibitor of cAMP-dependent protein kinase N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) (12.5-50 microM) decreased the rate of ovulation of the follicle-enclosed Rana temporaria oocytes induced by the homologous pituitary extract in amphibian Ringer solution and in a chloride-free medium. The inhibitor of voltage-dependent calcium channels diltiazem (10 and 100 microM) decreased the rate of ovulation in Ringer solution but did not affect it in a chloride-free medium or decreased the ovulation inhibitory effect of this medium. It was concluded that cAMP-dependent protein kinase and intracellular free calcium ions were involved as second messengers in the gonadotropin regulation not only in maturation of amphibian oocytes but also in ovulation.

[The effect of the composition of the medium on the concentration of free calcium ions in the cells of the follicular wall in the common frog and in the clawed toad]. / Vliianie sostava sredy na kontsentratsiiu svobodnykh ionov kal'tsiia v kletkakh stenki follikulov travianoi i shportsevoi liagushek.

The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.

Exclusively juvenile centrioles in Xenopus laevis oocytes injected with preparations of mature centrioles.

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of loach fish Misgurnus fossilis or from rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization is speculated.

[Effect of a chloride channel inhibitor and chloride-free medium on choriogonin-induced cAMP formation by Xenopus laevis follicles]. / Vliianie ingibitora khlornykh kanalov i ne soderzhashchei ionov khlora sredy na indutsiruemoe khoriogoninom obrazovanie follikulami shportsevoi liagushki tsAMF.

The inhibitor of chloride channels DIDS (10 microM) decreased and a chloride-free medium increased the cAMP level in the Xenopus laevis follicles stimulated by human chorionic gonadotropin. Also, DIDS inhibited while the chloride-free medium stimulated maturation of the follicle-enclosed oocytes.

Involvement of chloride channels in progesterone production during meiotic maturation of follicle-enclosed oocytes of Rana temporaria and Xenopus laevis.

The chloride channel blockers SITS (4-acetamido-4'-isothiocyanostibene-2,2'-disulphonic acid) and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) markedly suppressed progesterone production in the Rana temporaria and Xenopus laevis follicle-enclosed oocytes and oocyte maturation stimulated by the homologous pituitary suspension and hCG, respectively. Inhibition was dose-dependent and decreased with the increase of the hormone concentration. SITS did not affect progesterone production in the R. temporaria follicle-enclosed oocytes stimulated by dbcAMP. Substitution of sodium chloride for equimolar concentrations of sodium gluconate, methanesulfonate, glutamate, or formate significantly potentiated the gonadotropin-stimulated progesterone production and oocyte maturation in the both species. Possible involvement of chloride channels in progesterone production by the gonadotropin-stimulated amphibian follicle-enclosed oocytes is discussed.

[The role of eicosanoids and progesterone in the ovulation of the oocytes in the common frog]. / Rol' éikozanoidov i progesterona v ovuliatsii ootsitov travianoi liagushki.

Prostaglandin F2 alpha (1-5 micrograms/ml) stimulated ovulation in vitro of Rana temporaria oocytes in the absence of pituitary suspension and potentiated the effects of progesterone. The inhibitor of cyclooxygenase indomethacin (0.01-10 micrograms/ml) decreased the rate of oocyte ovulation stimulated by the pituitary suspension. An increased pituitary suspension concentration decreased the inhibitory effect of indomethacin. Indomethacin did not affect oocyte ovulation stimulated by prostaglandin F2 alpha or progesterone. The inhibition of ovulation by the chloride channel blocker SITS (10 microM) is partly relieved by prostaglandin F2 alpha or progesterone but completely eliminated by their mixture.