Influence of culture medium osmolality on maturation and ovulation of common frog oocytes stimulated in vitro by pituitary 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 μg/ml) was studied. During hibernation, 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 was reduced. 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.     

Intranuclear lipids in the late oocytes of the common frog

The complex of chromosomes and nucleoli, constituting the karyosphere with a capsule, was removed micro-surgically from the late oocyte nuclei of Rana temporaria. Lipids of nuclei and of karyosphere were investigated using biochemical and autoradiographical methods in hormone-stimulated maturing oocytes in vitro. Neutral lipids (triglyceride, diglyceride, cholesterol ester) were found in the karyosphere substance by thin-layer chromatography. During oocyte maturation the incorporation of a precursor (3H-glycerol) into triglyceride was seen to increase much more than into lecithin. The autoradiography on the sectioned oocytes showed that the intranuclear level of 3H-glycerol was more densely distributed in the nucleolar zone over the material of a fibrous component of the karyosphere capsule. The level was also detected over the central part of the karyosphere in close proximity to the chromosomes. The involvement of lipids in organization of the complicated intranuclear complex of the karyosphere with a capsule is discussed. It is suggested that lipid accumulation in the area of the karyosphere fibrous component may reflect their functional relation with the oocyte nuclear matrix.     

Intranuclear actin microfilaments in the oocytes of the common frog

For identification and distribution of actin microfilaments in hand-isolated nuclei of R. temporaria oocytes (stage 6, according to Dumont, 1972) different methods were used: heavy meromyosin decoration, antiactin immunofluorescence with monoclonal antibodies, staining with rhodamine phalloidin, and electrophoresis in polyacrylamide gel. The nuclei of R. temporaria oocytes contain a considerable quantities of actin microfilaments which form intranuclear meshwork. Microfilaments are connected with the nucleoli, nucleolar RNP-complexes and nuclear envelope. Immunofluorescence with antiactin monoclonal antibodies reveals a strong staining of microfilaments and nucleoli. A slight staining of nucleoli is observed after the treatment of nuclei with rhodamine phalloidin. A specific role of intranuclear microfilaments in direct transport of nucleolar material from the nucleus into the oocyte cytoplasm, in stabilization of the karyosphere (the late diplotene oocyte complex of chromosomes with numerous nucleoli) is discussed in addition to its keeping in a definite region of the nucleus. A supposition is drawn on the functional significance of the connection between microfilaments and nuclear matrix. Based on our own and literature data, a conclusion is drawn, that the intranuclear filament actin may be one of the leading components in morpho-functional organization of the nucleus as the whole.     

Refrigerated storage of European common frog Rana temporaria oocytes

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1–5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1–5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.     

Karyosphere of the oocytes of the common frog, Rana temporaria]

Karyospheres of ca. 200 mcm in diameter were isolated from the common frog oocytes of definitive size. An electron microscope study has revealed in the karyosphere fibrillar nucleoli and micronucleoli, modified synaptinemal complexes sometimes connected with chromatin and fibrillar material containing a great number of, mostly atypical, pore complexes resembling those of nuclear membrane and forming "pseudomembranes". An electrophoresis of the isolated karyosphere has revealed 12 distinct protein bands, of 3 which correspond to the protein triplet characteristic of the nuclear matrix and the rest 9 represent high molecular weight components with the molecular weight from 130 to 200,000 D.     

Glycogen synthesis by the direct or indirect pathways depends on glucose availability: in vivo studies in frog oocytes

Besides the classic direct route, frog oocytes incorporate glucosyl units into glycogen by the so-called indirect pathway. The operation of both pathways depends on glucose availability. Below 0.5 mM glucose (calculated intracellular concentration), the indirect route accounts for 90% of polysaccharide formation, while the direct pathway supports 70% of total glucose incorporation when administered glucose is above 1.5 mM. A sigmoidal curve was obtained for the direct pathway with n(H)=2.04, and half saturation was reached at 2.6 mM glucose. The curve for the indirect route presented an n(H) of 1.15 and an S(0.5) of 0.9 mM glucose.     

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 aquaporin inhibitors (mercuric chloride and methylmethanethiosulphonate) inhibited also homologous pituitary extract-induced volume increase in follicle-enclosed oocytes and led 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 of oocyte ovulation in the common frog caused an increase in their hydration that is necessary for their ovulation but this did not occur in denuded cells.     

Effects of gonadotropin-releasing hormone agonists on meiotic maturation of follicle-enclosed oocytes in rabbits

The effects of GnRH agonists on in vitro maturation of rabbit follicle-enclosed oocytes were studied. Rabbit preovulatory follicles were cultured with or without hCG (10(2) ng/ml), buserelin (10(2)-10(5) ng/ml), or leuprolide (10(2)-10(5) ng/ml) for 14 hours in vitro. GnRH agonists induced the resumption of meiosis in the follicle-enclosed oocytes in a dose-dependent manner. The percentage of oocytes achieving GVBD following treatment with 10(5) ng/ml buserelin (87.9 +/- 6.3%) or 10(5) ng/ml leuprolide (86.0 +/- 4.1%) did not differ significantly from hCG-treated control (87.3 +/- 3.8%). Mature oocytes initially were detected within 2 hours of GnRH agonist exposure. Concomitant addition of a GnRH antagonist at 10(4) ng/ml significantly blocked the stimulatory effect of GnRH agonist on oocyte maturation. GnRH agonists significantly stimulated both prostaglandin (PG) E2 (PGE2) and PGF2 alpha production by preovulatory follicles (p less than 0.01), but secreted prostanoid levels did not differ significantly among different concentrations of GnRH agonists. Meiotic maturation of follicle-enclosed oocytes following GnRH agonist exposure began 2 hours earlier than production of PGs. PG production stimulated by GnRH agonists was reduced significantly by indomethacin. However, oocyte maturity in the presence of GnRH agonist plus indomethacin did not differ significantly from that of GnRH agonist alone. GnRH agonistic analogues induce the resumption of meiosis in follicle-enclosed oocytes in rabbits by a mechanism other than PG stimulation.     

Successful cryopreservation of mouse oocytes by using low concentrations of trehalose and dimethylsulfoxide

Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.     

Sh and eag K(+) channel subunit interaction in frog oocytes depends on level and time of expression

Subcellular clustering of ion channels critically affects neuronal function. Coexpression of Eag and Sh channel subunits in Xenopus oocytes leads to accelerated decay of the Sh-like transient K(+) current (Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We report that such interaction depends critically on functional expression level (controlled by RNA injection quantities and indicated by current amplitudes) and developmental time after RNA injection. The accelerated decay became apparent 3 days after coinjection and increased thereafter. This was observed in different ionic conditions and at different voltage steps. However, decay was not accelerated at low expression levels, either within 1-2 days after injection or with reduced amounts of RNA. With sequential RNA injection, preformation of either Eag or Sh channels prevented interactions with the other subunit. The carboxyl terminus of Eag was found to be involved in accelerating, and in retarding recovery from, N-type inactivation. The interaction was reduced upon patch excision in macropatch measurements, suggesting involvement of cytosolic factors. We have reproduced the absence of interaction between Eag and Sh reported previously within 2 days after RNA injection and with low levels of current expression (Tang, C.-Y., C. T. Schulteis, R. M. Jiménez, and D. M. Papazian. 1998. Biophys. J. 75:1263-1270). Our findings demonstrate that heterologous expression of channels in Xenopus oocytes is a dynamic process influenced by cell physiology and development. These factors must be considered in interpreting the functional properties of heterologously expressed channels.     

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium is stimulated by low concentrations methane or methanol

Co-metabolic biodegradation of trichloroethylene by Methylosinus trichosporium OB3b was stimulated by low concentrations of methane (up to 70 M) or methanol (up to 0.4 mM) but inhibited at higher concentrations of them. A kinetic equation describing the dual effects of methane or methanol is proposed and the relevant kinetic constants have been determined.     

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.     

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes

Effect of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes was investigated using a fluorescent dye chlortetracycline. It is shown that in progesterone treated oocytes prolactin in concentration 50 ng/ml inhibits Ca2+ exit from intracellular stores of pig oocytes. Theophylline exerts the effect on prolactin Ca2+ exit from intracellular stores of pig oocytes. Employment of protein kinase C inhibitor cancelled inhibitory effect of prolactin and theophylline on Ca2+ exit from intracellular stores of pig oocytes. Ca2+ exit from intracellular stores of pig oocytes caused a joint influence of prolactin and GDP, and that of theophylline and GTP. The influence of protein kinase C inhibitor cancelled the stimulating effect of prolactin and GDP on Ca2+ exit from intracellular stores of pig oocytes also did not render any influence on the action of theophylline and GTP. These data suggest the influence of progesterone on theophylline and prolactin stimulated Ca2+ exit from intracellular stores of pig oocytes.     

Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.