Research Article
Detection of deoxyribonuclease I and II activities in Japanese quail oocytes
- Urszula Stepińska, Bozenna Olszańska
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- 17 May 2001, pp. 1-7
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Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using λ DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 × 10−3 Kunitz units and DNase II about 4 × 10−2 Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.
In vitro fertilisation of mouse oocytes reconstructed by transfer of metaphase II chromosomes results in live births
- Min-Kang Wang, Da-Yuan Chen, Ji-Long Lui, Guang-Peng Li, Qing-Yuan Sun
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- 17 May 2001, pp. 9-14
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The interaction between nucleus and cytoplasm can be explored through nuclear transfer. We describe here another tool to investigate this interaction: MII meiotic apparatus transfer (MAT) between mouse oocytes. In this study, the MII oocyte meiotic apparatus or spindle from C57BL/6 mice, a black strain, was transferred into an enucleated metaphase oocyte from Kunming mouse, a white strain. The results showed that the enucleation rate by treating oocytes with 3% sucrose was 100%, but the electrofusion efficiency was very low, with only 17.6% of reconstructed karyoplast-recipient cytoplasm pairs fused. When the fused oocytes were exposed to spermatozoa from C57BL/6 mice, 9 of 11 (82%) were fertilised. Eight reconstructed embryos at 1- to 4-cell stages were transferred into the oviducts of two synchronously pregnant Kunming strain fosters and one delivered two normal C57BL/6 offspring. This study indicates that MII meiotic apparatus or spindle sustains normal structure and function after micromanipulation and electrofusion. MAT provides a model for further research on the application of this technique to assisted human reproduction.
Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes
- Hideki Tatemoto, Norio Muto
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- 17 May 2001, pp. 15-23
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The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.
The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm
- A. Pavlok, M. Kubelka, J. Pěknicocá
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- 17 May 2001, pp. 25-38
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In this paper the effects of capacitation and fertilisation stimulating compounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS), bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed in several in vitro fertilisation protocols. Attention was paid to the rate of penetrated oocytes, kinetics of penetration and to polyspermic fertilisation. Cryopreserved bovine sperm and in vitro matured bovine oocytes were used throughout all the fertilisation experiments. As detected in the first 8 h fertilisation experiment with non-incubated sperm, the supplementation of medium with heparin, BOS and glucose supported the fertilisation rate most effectively (100%), including the kinetics of pronuclei formation (52.4%). The absence of BOS resulted in a decreased fertilisation rate (62.7%) as well as a delay in pronuclei formation (13.6%), similar to that after substitution of heparin with caffeine (73.0% and 25.4%, respectively). The penetration rate in the control medium with BOS (without heparin and caffeine) was surprisingly high, especially in medium without glucose (62.2%). The positive effect of glucose on sperm penetration was observed mainly in a chemically defined medium with PVA. High polyspermy rates were observed throughout all experiments in the media containing heparin or caffeine and BOS as the macromolecular component. D-Penicillamine was not shown to be a fertilisation-stimulating molecule. However, as detected in the second experiment in which oocytes were fertilised with 5 h incubated sperm, its positive effect on the prolongation of a fertile life span of cryopreserved spermatozoa was significant. The presence of either caffeine or heparin in the fertilisation medium (FM) with BOS during sperm incubation induced tyrosine phosphorylation of an approximately 90 kDa protein, detected after 5 h of sperm incubation. The absence of BOS reduced tyrosine phosphorylation of this protein in fertilisation medium with heparin. The percentage of motile spermatozoa and those with intact acrosomes were monitored throughout all experiments.
Expression and localisation of heat shock protein 70 in cultured bovine oocytes and embryos
- Sheldon J. Kawarsky, W. Allan King
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- 17 May 2001, pp. 39-50
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Effects of elevated in vitro temperature on in vitro produced early bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (hsp70). In vitro matured bovine oocytes, 2-cell and 8-cell embryos, and day 9 hatched blastocysts subjected to control and elevated temperature conditions were analysed by semiquantitative reverse transcription polymerase chain reaction methods for hsp70 mRNA expression. Results revealed the expression of hsp70 mRNA under control conditions and that early embryos can respond to heat stress by transcribing hsp70 mRNA. Confocal laser scanning microscopy used to localise the hsp70 protein in oocytes and embryos revealed that the distribution of hsp70 in the ooplasm of immature and mature oocytes is unaffected by exposure to elevated temperatures and that this protein was closely associated with the meiotic spindle, indicating its possible role in stabilising this structure. In 8-cell embryos derived under control conditions, hsp70 was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed to elevated temperature. In heat-stressed hatched blastocysts, a more even distribution was noted following heat stress relative to corresponding controls, indicating their competence to respond to elevated temperature.
Mammalian sperm molecules that are potentially important in interaction with female genital tract and egg vestments
- Daulat R.P. Tulsiani, Aida Abou-Haila
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- 17 May 2001, pp. 51-69
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Fertilisation is a highly programmed process by which two radically different cells, sperm and egg, unite to form a zygote, a cell with somatic chromosome numbers. Development of the zygote begins immediately after sperm and egg haploid pronuclei come together, pooling their chromosomes to form a single diploid nucleus with the parental genes. Mammalian fertilisation is the net result of a complex set of molecular events which allow the capacitated spermatozoa to recognise and bind to the egg's extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fuse with the egg plasma membrane. Sperm-zona (egg) interaction leading to fertilisation is a species-specific carbohydrate-mediated event which depends on glycan-recognising proteins (glycosyltransferases/glycosidases/lectin-like molecules) on sperm plasma membrane (receptors) and their complementary glycan units (ligands) on ZP. The receptor-ligand interaction event initiates a signal transduction pathway resulting in the exocytosis of acrosomal contents. The hydrolytic action of the sperm glycohydrolases and proteases released at the site of sperm-egg interaction, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of egg investments. This review focuses on sperm molecules believed to be important for the interaction with the female genital tract, passage through cumulus oophorus and attachment to ZP, induction of the acrosome reaction, secondary binding events, and passage through the ZP. An understanding of the expression and modifications of molecules thought to be important in multiple events leading to fertilisation will allow new strategies to block these modifications and alter sperm function.
A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes
- Stephen M. Downs
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- 17 May 2001, pp. 71-82
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This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 μl drops containing either 300 μM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18α-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 μM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP±FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4 and 5 h), meiotic induction was immediately blocked. In addition, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.
A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus
- Koji Nakagawa, Shuji Yamano, Hisayo Nakasaka, Kenji Hinokio, Midori Yoshizawa, Toshihiro Aono
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- 17 May 2001, pp. 83-88
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Parthenogenetic activation with various combinations of the calcium ionophore A23187 and protein synthesis or phosphorylation inhibitors was investigated as a means of producing human parthenogenones with one haploid pronucleus. Unfertilised human aged oocytes exposed to 5 μM A23187 for 5 min were treated with 10 μg/ml puromycin (puromycin group, 46 oocytes) or 2 mM 6-dimethylaminopurine (DMAP group, 42 oocytes) for 5 h. Oocytes treated only with A23187 served as a control (control group, 40 oocytes). After washing the oocytes, they were incubated for up to 37 h. Evidence of activation (pronuclear formation) and cleavage was observed 18 h and 42 h after A23187 treatment, respectively. Activation rates in the puromycin and DMAP groups were significantly higher than in the control group (91% (42/46) and 77% (34/44) vs 20% (8/40), p < 0.05, respectively). In the puromycin group, 81% (34/42) of the activated oocytes showed one pronucleus with the second polar body (2ndPB), whereas none (0/34) of the activated oocytes in the DMAP group extruded the 2ndPB. The cleavage rate in the puromycin group was significantly lower than in the DMAP group (38% vs 68%, p < 0.05). The activated oocytes which had one pronucleus with the 2ndPB in the puromycin group showed a haploid set of chromosomes (10/13). In conclusion, the combination of A23187 and puromycin is effective for producing human parthenogenones with one haploid pronucleus.
Activation of bovine oocytes matured in vitro by injection of bovine and human spermatozoa or their cytosolic fractions
- Osamu Okitsu, Shuji Yamano, Toshihiro Aono
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- 17 May 2001, pp. 89-95
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The aim of this study was to investigate whether bovine spermatozoa possess so-called sperm factor in the cytosolic fraction (CF) which activates bovine oocytes, and whether bovine oocytes matured in vitro are activated by microinjection of CF extracted from spermatozoa of other species. In the first experiment, bovine and human spermatozoa were microinjected into ooplasm of bovine oocytes matured in vitro. Secondly, CF from bovine and human spermatozoa were injected into bovine oocytes. In the third, CF from human spermatozoa was injected into human unfertilised oocytes obtained 18-20 h after clinical intracytoplasmic sperm injection (ICSI). We found that microinjection of bovine spermatozoa into bovine oocytes induced oocyte activation, as shown by resumption of meiosis and formation of a female pronucleus, at a significantly higher rate than the bovine sham injection (63.0% vs 43.0%; p < 0.05). On the other hand, there was no significant difference in activation rate between the human sperm injection (35.9%) and the human sham injection (22.9%). Furthermore, microinjection of bovine sperm CF into bovine oocytes induced oocyte activation at a significantly higher rate than the human CF injection or sham injection (75.9% vs 14.8%, 20.4%; p < 0.01). Formation of a single female pronucleus and second polar body extrusion was observed in 95.1% of activated oocytes after bovine sperm CF injection. When human sperm CF was injected into human unfertilised oocytes, the activation rate was significantly higher than following sham injection (76.9% vs 44.0%; p < 0.05). These results indicate the presence of sperm factor in bovine sperm CF which activate bovine oocytes, and suggest the possibility that sperm factor has species-specificity at least between bovine and human.