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.     

Role of protein kinase A and calcium ions in regulating ovulation of oocytes by gonadotropins in the grass frog (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.     

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.     

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.     

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.     

Role of prolactin in nuclear maturation and ovulation in amphibian oocytes

The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a significant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers.     

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.     

Effects of pH on in vitro ovulation of goldfish (Carassius auratus) oocytes

The in vitro effects of pH on goldfish (Carassius auratus) ovulation were investigated. Final oocyte maturation and follicular detachment were induced in vivo in gravid goldfish by HCG injections and elevated holding temperatures. Females were biopsied to determine the time of germinal vesicle breakdown and when oocytes should be removed for in vitro incubations. Prior to ovulation, the ovaries were removed, dissected and mature intrafollicular oocytes were incubated in media of varying pH (7.3-8.9). There was a significant increase in ovulation as the pH of the incubation increased and this ovulation could be blocked by indomethacin. Indomethacin did not inhibit the actual mechanism of oocyte expulsion since exogenous PGF2 alpha induced ovulation in indomethacin blocked incubates. Increased pH did not increase the ovulatory response observed with exogenous PGF2 alpha. The combined results suggest that an increase in incubation pH stimulates prostaglandin synthesis that, in turn, stimulates ovulation.     

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.     

排卵后老化卵母细胞的染色体形态变化

小鼠排卵后的卵母细胞停滞在MⅡ期, 如果此时的卵母细胞未能及时受精, 随着在输卵管中停留时间的延长, 卵母细胞会逐渐发生老化。这种卵母细胞的老化会导致包括人在内的哺乳动物的胚胎发育异常, 所以很有必要研究排卵后卵母细胞的老化机理。本实验主要研究小鼠排卵后的卵母细胞在体内老化过程中的染色体形态变化, 发现随着老化时间的延长, 有更高比例(65%, hCG后34 h)的卵母细胞的染色体呈不对称和松散的状态, 进一步研究表明, 这种染色体的变化可能与H3K14、H4K16的乙酰化升高, 及H3K9的甲基化降低有关。     

Role of glutathione in the maturation and fertilization of pig oocytes in vitro

The present study examined, by treatment of buthionine sulfoximine (BSO), which is a specific inhibitor of glutathione (GSH) synthesis, the role of GSH in the maturation and fertilization of pig oocytes in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were cultured for 36 h in Waymouth MB 752/1 with or without BSO (1 mM), fertilized in vitro, and assessed for GSH concentration (before insemination), maturation, and fertilization. The addition of BSO to maturation medium immediately after culture (Group I), 12 h after culture (Group II), or 24 h after culture (Group III) significantly decreased the GSH concentration in pig oocytes compared with the control (P < 0.01), whereas the rate of cumulus mass expansion at 36 h of culture and the rates of nuclear maturation and sperm penetration following in vitro insemination did not differ. However, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the control and Group III than in oocytes matured in Groups I and II (P < 0.01). In experiment 2, when BSO was added to maturation media 15, 18, 21, or 24 h after culture, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the medium supplemented with BSO at 21 or 24 h of culture than in oocytes matured in other groups (P < 0.05 or P < 0.01). The results indicate that GSH synthesis occurs throughout in vitro maturation of pig oocytes and GSH is an important cytoplasmic factor for regulating sperm nuclear decondensation and male pronucleus formation following sperm penetration in pig oocytes. © 1993 Wiley-Liss, Inc.     

Role of follicular cells in the inertia period of oocyte maturation in the common frog

Out of the breeding season the in vitro maturation of Rana temporaria oocytes in the state of maturation inertia or close to it depends on the follicular cells. In 28 females the presence of follicular cells stimulated oocyte maturation, in 12 females inhibited it. Dibutyrylcyclic AMP (5 X 10(-5) M) increased the percentage of maturation of follicle--enclosed oocytes close to the state of maturation inertia; estrone (4 X 10(-5)-10(-7) M and more) decreased the percentage of maturation of oocytes in the state of inertia, both with and without follicular envelopes.