Parthenogenetic Activation Of Oocytes Research Articles

Mouse in vitro fertilization, embryo development and viability, and human sperm motility in substances used for human sperm preparation for assisted reproduction

To investigate the endotoxin content and effects on mouse IVF and embryo development and human sperm motility of human sperm separation substances. One-cell and zona-free two-cell mouse embryo bioassays and Limulus Amoebocyte Lysate endotoxin tests and mouse oocyte parthenogenetic activation, IVF, preimplantation and postimplantation embryo development, and human sperm motility were performed in control medium or medium containing Percoll, Nicodenz, or the washings from Sperm Prep sephadex columns. Research Laboratories, Sinai Hospital of Baltimore, Baltimore, Maryland. Endotoxin levels, embryo development, and sperm motility. Mouse embryo bioassays indicated negative endotoxin levels in Percoll and Nicodenz but Limulus Amoebocyte Lysate assays had positive gel formation. Mouse IVF and preimplantation development was equivalent in control and Percoll- and Nicodenz-containing medium; none had parthenogenetic properties but postimplantation development was reduced for embryos grown in the presence of Percoll or Nicodenz. Human sperm remained motile in the presence of Percoll or Nicodenz. Sperm Prep column washes were toxic to mouse gametes and embryos and human sperm and had gel formation with the Limulus Amoebocyte Lysate assay. Percoll and Nicodenz, but not Sperm Prep sephadex column washes, were compatible with mouse preimplantation and postimplantation embryo development and human sperm motility.

The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte

The effect of temperature fluctuation on spindle integrity and chromosomal organisation in the human oocyte, and the consequences of such effects on the chromosomal constitution of resulting parthenotes, were investigated. A total of 340 oocytes were stained immunocytochemically with an antibody to alpha-tubulin, and 502 were activated parthenogenetically. Exposure of oocytes to room temperature for 2, 10 or 30 min caused disruption of the spindle in 77% (n = 26), 72% (n = 18) and 89% (n = 19) of cases respectively, with evidence of chromosomal dispersal in 50%, 56% and 52.6% respectively. These effects were reversed when oocytes were returned to 37 degrees C after exposure to room temperature for 2 min, but not after 10 min or 30 min. Temperature reduction affected rates of parthenogenetic activation of oocytes (2 min: 67%, n = 27; 10 min: 68%, n = 28; 30 min: 54%, n = 35) and cleavage of resulting parthenotes, but only if oocytes were exposed to room temperature for 30 min (30 min: 53%, n = 19). There is a direct association between temperature-induced spindle damage in the oocyte (70%, 50 of 63) and chromosomal abnormalities in parthenotes developed from oocytes exposed to room temperature (56%, 23 of 41; p < 0.01).

Ovarian teratomas in mice lacking the protooncogene c-mos.

Parthenogenesis has been suggested to be tightly coupled with development of ovarian teratomas. Indeed, ovarian tumors developed in c‐mos‐delieicnt female mice, which are characterized by the parthenogenetic activation of oocytes. The tumors appeared at a frequency of 30% between 4 and 8 months of age, and did not develop in younger or older mice. Most of the tumors were benign and consisted of multi‐focal cysts most notably with mature ectodermal components, but also with mesodermal and endodermal components. One among 17 tumors observed consisted of extraembryonic tissues alone, and two bore malignant components with metastasis to peritoneal organs. The results strongly suggest the involvement of c‐mos mutations in human germ cell tumors.

Parthenogenetic activation of oocytes in c-mos-deficient mice.

In Xenopus the c-mos proto-oncogene product (Mos) is essential for the initiation of oocyte maturation, for the progression from meiosis I to meiosis II and for the second meiotic metaphase arrest, acting as an essential component of the cytostatic factor CSF. Its function in mouse oocytes is unclear, however, as is the biological significance of c-mos mRNA expression in testes and several somatic tissues. We have generated c-mos-deficient mice by gene targeting in embryonic stem cells. These mice grew at the same rate as their wild-type counterparts and reproduction was normal in the males, but the fertility of the females was very low. The c-mos-deficient female mice developed ovarian teratomas at a high frequency. Oocytes from these females matured to the second meiotic metaphase both in vivo and in vitro, but were activated without fertilization. The results indicate that in mice Mos plays a role in the second meiotic metaphase arrest, but does not seem to be essential for the initiation of oocyte maturation, spermatogenesis or somatic cell cycle.

Removal of the zona pellucida and parthenogenetic activation affect rates of survival of ultrarapidly frozen mouse oocytes

Survival rates on thawing were assessed for ultrarapidly frozen mouse oocytes following the removal of zonae pellucidae using an acid Tyrode's solution, pronase or a mechanical dissection technique. Significantly higher rates of survival were observed for zona-free oocytes than for zona-intact control oocytes (303/684, 44% v. 130/498, 26%; P < 0.001). The rates of survival observed for pronuclear stage embryos (72-76%) were much greater than those observed for oocytes and were not influenced by zona removal techniques. Parthenogenetic activation of oocytes by exposure to a 7% (v/v) ethanol solution was also shown to increase survival rates of ultrarapidly frozen oocytes (155/185, 84% v. 19/102, 19%) indicating that fusion of sperm to the plasma membrane or formation of a male pronucleus are not directly responsible for the increased survival rates of pronuclear stage embryos compared with oocytes. These data support the hypothesis that increased survival of ultrarapidly frozen pronuclear stage embryos is the result of changes to the plasma membrane and/or to the zona pellucida that occur following fertilization.

Developmental ability of enucleated bovine oocytes matured in vitro after fusion with single blastomeres of eight-cell embryos matured and fertilized in vitro.

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment 1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26-28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polar body extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6-7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts.

Effects of Antisperm Antibodies on Early Cleavage of Fertilized Ova1

Early cleavage of zygotes was abnormal after oocytes were fertilized with sperm cells from one of 64 infertile couples studied in our human in vitro fertilization and embryo development procedure. The abnormal cleavage, which did not affect fertilization, was not due to polyspermy or parthenogenetic activation of oocytes, but to antisperm antibodies present on the sperm cells. These antibodies did not react with zona-intact or zona-free human oocytes, although they reacted with the acrosomal (strongly) and tail (weakly) regions of human sperm. The antibodies recognized a double band comprising two glycoproteins of 14 +/- 3 and 18 +/- 3 kDa and a protein band of 22 +/- 3 kDa on the Western blot of lithium diiodosalicylate-solubilized human sperm extract. Antisera were raised in rabbits against the double band (14 +/- 3- and 18 +/- 3-kDa antigens) and the 22 +/- 3-kDa protein. Passive transfer of affinity-purified human or rabbit immunoglobulins directed against the double band (14 +/- 3- and 18 +/- 3-kDa antigens), but not those directed against the 22 +/- 3-kDa protein, caused a significant inhibition of early cleavage of oocytes without affecting pronuclear formation in mice. These results suggest that the sperm surface antigens of 14 +/- 3 and 18 +/- 3 kDa may provide an extranuclear signal to oocytes to divide in mice and humans. These antigens may also find clinical applications in the management of immunoinfertility and in the development of an antisperm contraceptive vaccine for humans.

Vitrification of mouse oocytes: Improved rates of survival, fertilization, and development to blastocysts

Rall and Fahy's (1985) vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5-8 degrees C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.

Zona pellucida modifications in the mouse in the absence of oocyte activation

Mouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168-175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313-324, 1987). This paper shows that dimethylsulfoxide causes proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.

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)

Ovarian teratocarcinomas in LT/Sv mice carrying t‐mutations

Ovarian teratomas that result from parthenogenetic activation of oocytes provide a double tool for developmental genetics. First, they provide a way of measuring recombination between a gene and its centromere. Second, in the absence of crossing over there is the potential of producing tumors that are homozygous for genes that would be lethal in the course of in utero embryonic development. We have applied both aspects to several t-haplotypes containing different early acting t-lethal genes. In a study of 26 tumors, genotyped by Southern blot analysis of the major histocompatibility complex (MHC), we measured the distance between the centromere and the start of the t-complex as 5.6 +/- 2.3 cM. We found a marked deficiency of t-homozygous genotypes among the tumors we studied, although T/T genotypes formed teratomas at levels comparable to controls. None of the lethal t-haplotypes we studied permit homozygous embryos to develop to the primitive streak stage, while T/T embryos do develop essentially normally through that stage. Thus, although the total number of tumors observed from t-bearing mice was small, the great difference in the incidence of t/t tumors versus the incidence of T/T tumors suggests strongly that the parthenogenetic embryos that convert to teratocarcinomas must first pass through some of the stages of normal early development, including the formation of three germ layers and the primitive streak.

Hormone-induced parthenogenetic activation of mature starfish oocytes

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca 2+ at that time, although it is shown that Ca 2+ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation. It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.