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At least 19 recordsLinked to original sources

PubMed1987

Inhibition of progesterone-mediated maturation of oocytes of Xenopus laevis by oocyte maturation inhibitor from pig follicular fluid: development of a routine assay for the inhibitor with Xenopus oocytes.

We describe an assay for oocyte maturation inhibitor (OMI) using the progesterone-mediated maturation of Xenopus oocytes. The test fraction used was a partially purified fraction from pig follicular fluid, which gave consistent inhibition of maturation in the pig oocyte assay. In the toad assay, oocytes (30-50) from each toad were pretested to determine whether satisfactory maturation was achieved because widespread animal variation was observed. Toads whose oocytes showed greater than 60% maturation in the pretest could be used directly. Toads whose oocytes showed less than 60% maturation were injected with pregnant mares serum gonadotropin (PMSG) in order to increase progesterone-mediated maturation. The dose and time after injection of PMSG before harvesting oocytes, dose and duration of progesterone exposure, and order of exposure of oocytes to OMI and progesterone were important variables. Opposing effects of OMI and progesterone were seen in oocytes from toads receiving 60 IU PMSG. In the routine assay we use animals whose oocytes show greater than 60% maturation in the pretest or animals treated with 12 IU of PMSG 4 to 7 days before use. Oocytes are exposed to the OMI fraction for 1 hr, progesterone is added, and incubations continued until controls reach maximum maturation (5 to 8 hr). The inhibition of toad oocyte maturation by OMI is reversible. The toad and mammalian oocyte assays were compared using more highly purified fractions of OMI and gave identical results.

Animals↗

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↗

PubMed1984

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.

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↗

PubMed1989

Hamster oocyte penetration tests with oocytes frozen in propanediol: comparison with non-frozen oocytes.

Hamster oocytes were frozen using a 1,2-propanediol-sucrose procedure, which resulted in over 90% survival. After thawing and zona removal the oocytes were compared with non-frozen oocytes in a zona-free hamster egg test employing spermatozoa from human semen donors and suspected infertility patients. Similar data were obtained, indicating that propanediol-sucrose frozen hamster eggs may be used in place of fresh eggs for convenience and to avoid scheduling problems.

Animals↗

PubMed1987

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation.

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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↗

PubMed1988

Chromosome analysis of preovulatory human oocytes and oocytes failing to cleave following insemination in vitro.

These investigations were carried out within a clinical in vitro fertilization and egg replacement program. A gradual fixation method for chromosome preparations of animal oocytes, when modified and applied to human oocytes, gave numerical analyzability in about 80%. Of preovulatory oocytes recovered in stimulated cycles, 53% of oocytes with numbers of chromosomes in the haploid range had an apparently normal set of 23 chromosomes. Oocytes with a very low number of chromosomes or in first meiosis were also found. In a separate investigation the chromosome constitution of oocytes not showing clinical signs of fertilization 48 hours after insemination in vitro was also studied. Of the oocytes with numbers of chromosomes in the haploid range, 50% had a normal 23,X karyotype. A different distribution of abnormal chromosome preparations was found, compared with the preovulatory oocytes. Twelve oocytes (16%) carrying between 10 and 18 chromosomes were found among the inseminated oocytes, but none in the other study. Moreover, 15 preparations displayed no chromosomes in the inseminated oocytes, whereas in the uninseminated group, none lacked chromosomes completely. These results may indicate how unfertilized human oocytes in vitro undergo chromosome loss and degeneration and could represent different stages of degeneration. Among both preovulatory and unfertilized oocytes the distribution between hyper- and hypohaploid oocytes was of particular interest in that hypohaploid chromosome complements were in excess. This finding supports the hypothesis that aberration in the number of chromosomes in an oocyte is caused not only by nondisjunction but also by anaphase lag, since nondisjunction alone would result in a 1: 1 ratio between hyper- and hypohaploid complements. In conclusion, human oocytes recovered for in vitro fertilization seem to have an incidence of numerical chromosome abnormalities in the region of 40-50%. This is close to the figures estimated on the basis of observations made in spontaneous abortions and could be a major explanation for the low developmental potential of human cleavage stage ova.

Cell Division↗

PubMed1988

Regulation of oocyte maturation: communication in the rat cumulus-oocyte complex.

This study was undertaken to analyse the time relationships between breakdown of communication in the cumulus-oocyte complex and reinitiation of meiosis in the oocyte. In addition the possibility was examined that under conditions of established communication, cAMP is transferred from the cumulus cells to the oocyte. Coupling in the cumulus-oocyte complex and maturation of the oocyte were examined, both in vitro in follicles exposed to LH and in vivo following injection of the hormone to immature, pregnant mare's serum gonadotrophin-primed rats. Transfer analysis was performed by determination of cAMP content in cumulus-enclosed as compared to cumulus-free oocytes incubated with forskolin. It was discovered that after 1 h of culture in the presence of LH, coupling in the cumulus-oocyte complexes had decreased to 45% of its initial level, while only 10% of the oocytes had resumed meiosis by this time. The decrease in coupling in cumulus-oocyte complexes in vivo was 20% by 2 h after administration of the hormone, with all the oocytes being immature at this time. Cyclic AMP determinations revealed that cumulus-enclosed oocytes contained concentrations of cAMP three-fold higher than cumulus-free oocytes incubated under similar conditions. This study demonstrates that, in the rat, (i) a decrease in coupling precedes reinitiation of meiosis and (ii) in the presence of established communication, cAMP is transferred from the cumulus cells to the oocyte. These findings suggest that a decrease in communication can serve as the signal for oocyte maturation, possibly by preventing transfer of cAMP from the cumulus cells to the oocyte.

Animals↗

PubMed1983

Regulation of mouse oocyte meiotic maturation: implication of a decrease in oocyte cAMP and protein dephosphorylation in commitment to resume meiosis.

Mouse oocytes are reversibly inhibited from resuming meiotic maturation in vitro by cAMP phosphodiesterase inhibitors such as 3-isobutyl-1-methyl xanthine (IBMX) and cAMP analogs such as dibutyryl cAMP (dbcAMP). Oocytes cultured in IBMX-containing medium were transferred to and cultured in IBMX-free medium for various periods of time prior to their return to either IBMX- or dbcAMP-containing medium. Results from these experiments defined a period of time in which oocytes became committed to resuming meiosis. Forskolin, which elevated the intracellular oocyte cAMP concentration, transiently inhibited oocytes from resuming meiosis. Levels of cAMP were determined in oocytes incubated in medium that allows resumption of meiosis. The level of oocyte cAMP decreased significantly during the time in which oocytes become committed to resuming meiosis. This decrease in oocyte cAMP was not observed in oocytes inhibited from resuming meiosis by IBMX. In addition, cAMP levels were determined in preovulatory antral follicles, cumulus cell-oocyte complexes, and oocytes during gonadotropin-induced resumption of meiosis in vivo. A decrease in oocyte cAMP preceded resumption of meiosis as manifested by germinal vesicle breakdown (GVBD). This decrease apparently occurred before or during a period of time in which follicle and cumulus cell cAMP were increasing. Associated with commitment to resume meiosis was a characteristic set of changes in oocyte phosphoprotein metabolism that preceded GVBD. These changes are, to date, some of the first reported biochemical changes that precede GVBD. Results from these experiments are discussed in terms of a possible role cAMP may play in regulation of resumption of meiosis in mammals.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes.

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.

Animals↗

PubMed1983

Decline of follicular oocyte maturation inhibitor coincident with maturation and achievement of fertilizability of oocytes recovered at midcycle of gonadotropin-treated women.

To examine whether a decline in follicular oocyte maturation inhibitor (OMI) is associated with attainment of oocyte maturation and fertilizability, OMI was measured in follicular fluid (FF) of 39 follicles of 20 normal women given human menopausal gonadotrophin and human chorionic gonadotrophin to induce follicular growth and maturation. Oocytes were aspirated per laparoscope, the fluid was saved, and the egg was observed, incubated, and inseminated with the husband's sperm. Concepti that developed to the 4- to 8-cell stage were transferred to the uterus and the women were followed for pregnancy. OMI activity in each FF was measured by using cultured cumulus-enclosed porcine oocytes (30-40 oocytes per FF sample). Estrogen, progesterone, oocytes (30-40 oocytes per FF sample). Estrogen, progesterone, and delta 4-androstenedione were measured in FF by radioimmunoassay. The FF of 13 preovulatory follicles yielding oocytes that were mature and fertilizable had significantly less OMI activity (mean +/- SEM) (0.58 +/- 0.10 unit/ml) compared to follicles yielding immature oocytes (2.8 +/- 0.56 units/ml; n = 9), atretic oocytes (5.5 +/- 2.5 units/ml; n = 7), or preovulatory oocytes with fractured zonae (1.9 +/- 0.63 units/ml; n = 7). The estrogen concentration (mean +/- SEM) of preovulatory follicles yielding mature fertilizable eggs or mature eggs with fractured zonae was greater (396 +/- 34 ng/ml; n = 20) compared to follicles yielding immature or atretic eggs (203 +/- 59 ng/ml; n = 9 and 97 +/- 47 ng/ml; n = 7, respectively; P less than 0.05). Progesterone concentration (mean +/- SEM; ng/ml) of FF was generally elevated in all preovulatory follicles (635 +/- 53) compared to immature or atretic follicles (230 +/- 64 and 76 +/- 17, respectively; P less than 0.05). It may be concluded that in normal follicle maturation there is a decline in OMI in the follicle containing an oocyte that becomes mature and fertilizable. There is also an increase in estrogen, progesterone, and follicle size. It is also possible to have an abnormal follicle maturation when there is an increase in size as well as FF, estrogen, and progesterone, but withut a decline in OMI--a situation which can lead to production of a nonfertilizable oocyte.

Adult↗

PubMed1994

Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection.

Knowledge of the timing of the stages of fertilization in humans is still limited because the time of gamete fusion is not known when pre-ovulatory or in-vitro matured cumulus-enclosed oocytes are inseminated. We therefore studied the morphological nuclear changes in 14 patients' oocytes by means of light microscopic observation at 2, 4, 6, 8, 16, 18 and at 20 h after intracytoplasmic single sperm injection (ICSI). A total of 144 metaphase II oocytes were injected with the spermatozoa of the patients' partners. Out of the 134 oocytes that survived the injection, 93 displayed two pronuclei in the course of the observation period (69%). Out of the 93 normally fertilized oocytes, 21 extruded the second polar body at 2 h after micro-injection (23%) and 63 oocytes at 4 h (68%). Pronuclei appeared as early as 6 h after ICSI in 16 normally fertilized oocytes (17%). At 8 h, 75 (80%) oocytes had two visible pronuclei, at 16 h 92 (99%), at 18 h 76 (82%) and at 20 h 63 (68%). In 24 oocytes (26%) the appearance of pronuclei was asynchronous, while the disappearance of the pronuclei was always synchronous, except in one oocyte. Nine of the 134 successfully injected oocytes showed three equal-sized pronuclei (6.7%). Four of the nine multi-pronucleated oocytes did not extrude the second polar body at all, while the time sequence of appearance of pronuclei was similar to that of the normally fertilized oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Follicular factors influence oocyte fertilizability by modulating the intercellular cooperation between cumulus cells and oocyte.

In order to investigate whether the follicular tissue influences cumulus-oocyte interaction and, consequently, the fertilizability of the egg, four experiments were carried out. In the first, cumulus-enclosed pig oocytes were cultured for 44 h in control medium (modified TCM-199) or in follicle-conditioned medium, and the intercellular coupling was studied by measuring 3H-uridine uptake. In control medium the intercellular cooperation started to decline immediately, and at 24-32 h the uncoupling was almost complete. By contrast, in follicle, conditioned medium, it remained at high levels until 24-32 h. In the second experiment protein synthesis patterns of oocytes were studied. Oocytes cultured in conditioned medium were characterized by a 45-kD protein band, while those maturing in control medium were identifiable by a marked 56-kD band. In the third experiment mature oocytes were fertilized in vitro. The percentage of penetrated egg was higher in oocytes matured in conditioned medium than in control medium. In addition, only oocytes matured in conditioned medium could consistently decondense spermatozoa and form male pronuclei. Metabolic cooperation, protein synthesis patterns, and fertilizability were also studied in oocytes matured in control medium supplemented with either 17 beta-estradiol or progesterone or testosterone or dihydrotestosterone or androstenedione or ether extract of conditioned medium. Only ether extract and progesterone stimulated cumulus oocyte interaction and sperm decondensation. In the last experiment oocytes denuded at different stage of their maturation in conditioned medium were fertilized in vitro. The longer the eggs were cultured with the cumulus, the higher was their penetrability. Moreover, only oocytes denuded after 40 h of culture could, once fertilized, promote the formation of male pronuclei. These data demonstrate that follicular secretions are fundamental for the maintenance in vitro of a functional intercellular coupling between cumulus and oocyte, which is necessary for the egg to become penetrable by spermatozoa and to acquire the conditions required for the formation of male pronuclei.

Animals↗

PubMed1989

Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age, and cumulus removal.

Cumulus-intact and -denuded unfertilized oocytes from two mouse strains were exposed to 1.5 M ethanol (EtOH) or two cryoprotectant solutions, 1.5 M propanediol (PROH) or 1.5 M dimethylsulfoxide (DMSO), for 4.5 min at 27 degrees C, and the proportion of activating or degenerating oocytes studied. Exposure to DMSO did not significantly increase activation above that of oocytes not exposed to DMSO. Treatment of oocytes in PROH resulted in the activation of up to 87% of viable oocytes. This was significantly higher (P less than .01) than in control oocytes and comparable to the rate of activation after treatment with EtOH (59-96% activation). In solutions at 1 degree C, 47% of control oocytes were activated, which was not significantly different from the rate of activation in EtOH (36%) or PROH (50%) at 1 degree C. Following treatment with PROH, up to 87% of oocytes degenerated within a period of 6 h in vitro. The age of the oocytes (h post hCG) and the time of cumulus removal with the enzyme hyaluronidase, relative to the time of exposure to the chemicals, influenced the level of degeneration in most groups. Significantly fewer oocytes degenerated when cumulus cells were removed before treatment (0-31%) than when the cumulus was left intact throughout the treatment and 6 h culture period (10-87%). Exposure to PROH at 1 degree C reduced oocyte degeneration to 5%. We conclude that PROH causes significantly greater losses of oocytes as a result of parthenogenetic activation and degeneration than of exposure to DMSO.

Animals↗

PubMed1986

Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes.

Three experiments were conducted for evaluation of the efficacy of conventional freezing or vitrification of hamster oocytes for use in a human sperm penetration assay (hSPA). In experiment 1, oocytes were cryopreserved and evaluated for survival on the basis of morphologic criteria. Survival of vitrified oocytes and that of frozen oocytes were not different, whereas all cryopreserved groups had lower survival than noncryopreserved controls. In experiment 2, oocytes were conventionally frozen or vitrified and evaluated in an hSPA. Vitrified oocytes had a lower frequency of sperm penetration than frozen oocytes, and all cryopreserved groups had lower penetration rates than untreated controls. In experiment 3, oocytes were exposed to the cryoprotectant used to vitrify (VS1) or freeze (DMSO) but not cooled prior to evaluation in an hSPA. Exposure to DMSO but not VS1 reduced hSPA values. It is concluded from these experiments that while all cryopreserved oocytes do not survive, at current stages of development conventionally frozen oocytes perform better than vitrified oocytes in the hSPA and losses associated with conventional freezing procedures may be related to cryoprotectant exposure, whereas vitrification losses are more probably due to events associated with rapid cooling and/or warming of the oocytes.

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↗