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At least 19 recordsLinked to original sources

PubMed1975

Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins.

Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.

Animals↗

PubMed1979

A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro.

Evidence is presented that strongly supports the hypothesis that the junctional association between oocytes and granulosa cells must be maintained to promote oocyte growth and development in vitro and that the coculture of oocytes with granulosa cells is not a sufficient condition for oocyte development. Furthermore, it is shown that incorporation of uridine and leucine by oocytes into TCA-insoluble material is significantly greater in granulosa cell-enclosed oocytes than in cocultured oocytes.

Animals↗

PubMed1990

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation.

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.

Animals↗

PubMed1991

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling.

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1 x 10(4) sperm/ml (P less than 0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zona-free oocytes had high rates of polyploidy (greater than or equal to 40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1 x 10(4)/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1 x 10(5) and 1 x 10(4) sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Control of chromosome behavior in amphibian oocytes. II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosome condensation in transplanted brain nuclei by oocyte cytoplasm.

We studied the effects of actinomycin D, alpha-amanitin, puromycin, and cycloheximide on the cytoplasmic activity of maturing Rana pipiens oocytes that induces chromosome condensation in transplanted brain nuclei. Treatment of oocytes with each inhibitor suppressed the chromosome condensation induced by metaphase oocytes to varying degrees depending upon the dose of inhibitor, despite the fact that untreated metaphase I oocytes already possessed chromosome condensation activity (CCA). Treatment of brain nuclei before injection completely suppressed condensation at all doses used. Chromosome condensation induced by metaphase II oocyte cytoplasm, however, was insensitive to all the inhibitors, even when the brain nuclei were pretreated. Oocytes treated with alpha-amanitin throughout maturation induced chromosome condensation when tested at metaphase II. Removal of the oocyte chromosomes after the germinal vesicle (GV) broke down did not prevent the development of CCA, whereas removal of the entire GV before initiation of maturation deprived oocytes of CCA. The results suggest that metaphase I oocyte cytoplasm stimulates synthesis of brain nuclear RNAs that are translated into proteins necessary for chromosome condensation, whereas metaphase II oocytes possess all the factors for chromosome condensation. In both cases, GV nucleoplasm appears indispensable for the development of CCA, whereas immediate activity of the oocyte genome is not required.

Amanitins↗

PubMed1990

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.

Animals↗

PubMed1991

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.

Adult↗

PubMed1990

Expression and translocation of cloned human estrogen receptor in the Xenopus oocyte does not induce expression of the endogenous oocyte vitellogenin genes.

Estrogen-receptor complex activates the genes coding for the egg yolk protein precursor vitellogenin in hepatocytes of oviparous vertebrates, while oocyte vitellogenin genes are unresponsive to the hormone. Localization of [3H]steroid hormones (estradiol, progesterone, testosterone, and dexamethasone) was assayed in 10% trichloroacetic acid-precipitated dissected Xenopus oocytes (germinal vesicle vs. the rest of the oocyte). Whether hormones were introduced by incubation in the medium surrounding the oocytes or by injection of an equivalent amount into the oocyte cytoplasm, all hormones partitioned into the nucleus at equivalent levels (approximately 5%), reflecting that portion of the oocyte volume occupied by its nucleus. Therefore, intracellular receptors for these hormones were not detectable. Subsequently, we introduced a species-heterologous estrogen receptor into the Xenopus oocyte via a recombinant plasmid containing the coding sequence for the human estrogen receptor (HER) housed in a vector that ensures highly efficient transcription and translation of inserted sequences. HER synthesis was directed from injected plasmid (2 ng/oocyte germinal vesicle) as shown by [35S]methionine incorporation into newly synthesized proteins; however, vitellogenin was not synthesized under these conditions. When HER plasmid-injected oocytes were incubated in [3H]estradiol, they translocated to the nucleus 38% of the radiolabeled estradiol taken up by the cells, compared to 5% nuclear localization for vector-injected controls. Therefore, although the oocyte can readily transcribe and translate HER sequences as well as appropriately partition the completed protein in the nuclear compartment, the endogenous, potentially estrogen-responsive vitellogenin genes of the oocyte are not expressed.

Animals↗

PubMed1992

Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size.

Development of mammalian oocytes is usually correlated with ovarian follicular development. This correlation was tested by determining whether gonadotrophic stimulation of follicular development in immature mice resulted in a coordinated increase in the embryonic developmental capacity of the oocytes. Oocyte cumulus cell complexes were isolated at the germinal vesicle stage from small, medium and large antral follicles of 26-day-old mice and matured and fertilized in vitro. The frequency with which embryos from oocytes from small follicles completed the two-cell to blastocyst transition was lower than for embryos from oocytes from large follicles (33% and 79%, respectively). Germinal-vesicle stage oocyte-cumulus cell complexes were isolated from 22-26-day-old mice that were unprimed or primed by injection of equine chorionic gonadotrophin 48 h before isolation. Oocytes were matured in control medium, or in medium containing 1 microgram follicle-stimulating hormone (FSH) ml-1, and then fertilized in vitro. Priming did not increase the number of embryos completing the two-cell stage to blastocyst transition in the 22-day-old group nor did FSH treatment of maturing oocytes when the oocytes were isolated from unprimed 22-day-old mice. In contrast, priming increased the percentage of embryos completing the two-cell stage to blastocyst transition in the 26-day-old group by 20%. FSH treatment of maturing oocytes from the unprimed, 26-day-old group increased the number of embryos completing the transition to the same level as those in the primed 26-day-old group, but FSH did not increase the frequency of transition in the primed 26-day-old group.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1991

Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells.

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

Animals↗

PubMed1992

Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte.

The volumetric response of oocytes during rapid alterations of the extracellular osmotic environment were recorded using video microscopy. From these observations, the kinetics of water loss for human and mouse oocytes were determined over the temperature range 37 to 10 degrees C, including 37, 30, 20, and 10 degrees C. The changes in diameter of oocytes were measured over a 5-min period and a computer model was used to derive values for membrane water permeability (Lp) and inactive volume (Vb) and to compare the experimental data to the predicted values. The results for the mouse oocyte Lp were comparable to values determined by other methods. However the human data, for both failed-to-fertilize and fresh oocytes, have a wide range of values with large standard deviations. The Lp values at the various temperatures were used to calculate the Arrhenius activation energy (Ea). An Ea value of 9.48 kcal/mol was found for the fresh mouse oocyte, whereas the activation energy for human oocytes was extremely low, 3.73 kcal/mol for fresh oocytes and 1.93 kcal/mol for failed-to-fertilize oocytes.

Animals↗

PubMedDate not supplied

Relationship of a human oocyte scoring system to oocyte maturity and fertilizing capacity.

A simple, quick, and semiquantitative human oocyte scoring system is described. Oocyte-corona-cumulus complexes were spread, either by tilting the dish or by aspiration of the fluid, and examined under the dissecting microscope. A maximum of four points was assigned to each of the following: cumulus expansion, cumulus appearance, amount of cumulus, corona expansion, corona appearance, and oocyte appearance. The oocyte score was significantly correlated to two important physiological parameters of oocytes, nuclear maturity (P less than .02) and fertilization rate (P less than .0001). This oocyte scoring system is useful for selecting oocytes for in vitro fertilization and embryo transfer (IVF-ET) or gamete intrafallopian tube transfer (GIFT), for training new laboratory personnel in recognizing the important characteristics of a mature oocyte, and in the standardized reporting of oocyte quality by different IVF-ET or GIFT programs.

Clomiphene↗

PubMed1992

A prospective study comparing the outcome of oocytes retrieved in the aspirate with those retrieved in the flush during transvaginal ultrasound directed oocyte recovery for in-vitro fertilization.

OBJECTIVE: To study prospectively the fate of oocytes collected from the follicular aspirate and subsequent flushes during transvaginal ultrasound directed oocyte recovery for in-vitro fertilization (IVF). SETTING: A tertiary referral assisted conception centre. SUBJECTS: 100 consecutive patients undergoing 100 cycles of IVF. Four patients were withdrawn because their embryos were electively cryopreserved. Therefore 96 cycles were studied. MAIN OUTCOME MEASURES: The oocyte recovery rate, viability, fertilization and cleavage rates and outcome of embryos generated from oocytes that were obtained from either the initial aspirate (A1), dead space in the collecting system (A2) or the first two 2 ml flushes (F1 and F2) were compared. RESULTS: The overall oocyte recovery rate was 87.8%. Of the 1046 oocytes collected, 40.3% were from A1, 41.3% from A2, 13.7% from F1 and 4.7% from F2. There were comparable numbers of viable and fertilized oocytes and cleaved, transferred and frozen embryos in tubes A1 and A2 but all these parameters were significantly lower in tubes F1 and F2 (P < 0.0001). All these parameters were also significantly higher in F1 compared with F2 (P < 0.001), except for the number of embryos frozen, in which there was no difference. The overall pregnancy rate per cycle was 28.1% and the pregnancy rate per embryo transfer was 31.0%. There were no pregnancies in any of the cycles in which embryos originating from F2 were transferred, nor were there pregnancies in cycles in which only embryos from F1 were transferred. CONCLUSION: Follicular aspiration together with one 2 ml flush maximises the recovery of oocytes that will result in pregnancies.

Adult↗

PubMed1978

Meiotic maturation of mouse oocytes in vitro: protein synthesis in nucleate and anucleate oocyte fragments.

Nucleate and anucleate fragments of mouse oocytes have been isolated following treatment of fully grown oocytes with cytochalasin B. The nucleate oocyte fragments resume meiosis in vitro, progressing from dictyate of the first meiotic prophase to metaphase II ('meiotic maturation'), and exhibit all of the changes in protein synthesis normally associated with meiotic maturation of mouse oocytes. The anucleate oocyte fragments also undergo certain of the changes in protein synthesis associated with meiotic maturation, despite the absence of nuclear progression. These results suggest that the acquisition of meiotic competence (i.e. the ability to undergo meiotic maturation) during growth of the mammalian oocyte is due to changes in the quality, rather than the quantity, of cytoplasm and that the reprogramming of protein synthesis during meiotic maturation is directed by RNA templates already present in the cytoplasm. The behaviour of anucleate oocyte fragments is discussed in terms of the proposed role for nucleoplasm in the initiation of changes in protein synthesis during meiotic maturation of mouse oocytes.

Animals↗

PubMed1990

[Recovery and light microscopic evaluation of follicular oocytes of swine and relationship between the degeneration rate of oocytes and the estrus phase].

Cumulus-oocyte complexes were recovered from 25 gilts by aspiration of follicular fluid or cutting of follicles from all Graafian follicles of greater than or equal to 3 mm in diameter during diestrus, proestrus or estrus. In 5 gilts the oocytes were collected post ovulation by flushing of oviducts. The recovery rate of follicular oocytes differed between 75.5% during the late diestrus (days 13-17) and 43.5% during the proestrus (days 18-21). During the proestrus and on day 1 of the estrus the recovery of oocytes was more difficult as a result of the higher viscosity of follicular fluid and the mucification of cumulus-oocyte complexes. The degeneration rate of oocytes was high during the diestrus with a peak at the time of regression of corpora lutea. From diestrus to the estrus the degeneration rate decreased. Following degeneration rates were found in the oocytes during the cycle: days 7-12: 38.8%, days 13-17: 50.0%, days 18-21: 29.6%, day 1 of the estrus: 10.8%, day 2 of the estrus ante ovulation: 11.8%, day 2 of the estrus post ovulation: 6.2%. Signs of degeneration were: Loss of cumulus cells (during diestrus and proestrus), damaged zona pellucida, enlargement of perivitelline space, deformation of oocyte, alteration of structure of the ooplasm, diameter of vitellus less than 100 microns. It was concluded that the selection of dominant follicles takes place in pigs during a long time of the cycle, especially during the diestrus. There were not any indications of a 2-wave hypothesis of follicular growth during the cycle in pig.

Animals↗

PubMed1978

Studies on oocyte maturation of the medaka, Oryzias latipes. VI. Relationship between the circadian cycle of oocyte maturation and activity of the pituitary gland.

The relationship between pituitary activity and oocyte maturation was examined in Oryzias latipes (medaka), which has a circadian cycle of oviposition. Throughout the circadian cycle of oviposition, females possessed a population of large oocytes more than 800 micronmeter in diameter that could mature in the presence of gonadotropin. Oocyte maturation was observed in vitro in females hypophysectomized between three and ten hours after the beginning of the light period with the number of maturing oocytes increasing as hypophysectomy was delayed. Although in vivo oocyte maturation was blocked by hypophysectomy within two hours after the beginning of the light period, it was restored by a single injection of synthetic or mammalian pituitary hormones (gonadotropic, corticotropic and thyrotropic hormones) within ten hours after hypophysectomy. Of these pituitary hormones, FSH, LH and TSH could induce in vitro maturation of isolated oocytes. Oocytes matured in vitro in the absence of exogeneous hormones if they were isolated nine or more hours after the onset of light. The present study indicates that the circadian cycle of maturation of Oryzias oocytes is controlled by the release of pituitary hormone between three and nine hours after the beginning of the light period.

Animals↗

PubMed1976

Analysis of mouse oogenesis in vitro. Oocyte isolation and the utilization of exogenous energy sources by growing oocytes.

A method is described for the dissociation of mouse ovaries and the isolation of oocytes free of somatic cells by agitating pieces of ovary in collagenase and deoxyribonuclease in a calcium and magnesium free salt solution. This method yielded about 50% of the growing oocytes from immature mice. The utilization of exogenously administered 14C-labelled energy sources by oocytes in various growth stages was determined by measurement of evolved 14CO2. Little or no evolution of 14CO2 was detected from oocytes of any size incubated in 14C-glucose, lactate or succinate. The production of 14CO2 from 14C-pyruvate increased logarithmically when plotted against increasing oocyte volume with a plateau occurring after occytes reached a volume of 65,500 mum3 (50 mum diameter). Thus, the pattern of energy metabolism for oocyte maturation and early egg cleavage, wherein glucose and lactate are not utilized as efficiently as pyruvate, has been established by the earliest stages of oocyte growth.

Animals↗

PubMed1991

Human preovulatory oocytes have a higher sperm-binding ability than immature oocytes under hemizona assay conditions: evidence supporting the concept of "zona maturation".

OBJECTIVE: The purpose of this study was to investigate the sperm-binding potential of human oocytes at different stages of nuclear maturation under hemizona assay (HZA) conditions. DESIGN: This was a prospective study designed in a blinded fashion. SETTING: Academic research environment approved by the Institutional Review Board. PATIENTS: Surplus oocytes, donated by patients undergoing in vitro fertilization therapy after gonadotropin stimulation, were analyzed. Semen from a fertile donor was used in all assays. INTERVENTIONS: Five groups of oocytes were considered: (1) immature, prophase I; (2) metaphase I; (3) metaphase II; (4) inseminated, unfertilized metaphase II; and (5) immature, prophase I oocytes matured in vitro to metaphase II. Oocytes were stored in salt solution (pH 7.2) and microbisected before assay. MAIN OUTCOME MEASURE(S): Tight binding of sperm to the zona pellucida under HZA conditions was evaluated after 4 hours of gametes coincubation. RESULTS: Metaphase II oocytes (groups 3 and 4) had significantly higher binding than other groups (P = 0.0001). The mean value of the difference between the two halves (hemizona) was not significant, thus showing a small intra-assay variation for all maturational stages. CONCLUSIONS: Full meiotic competence of human oocytes is associated with an increased zona pellucida-binding potential.

Double-Blind Method↗