Protein synthesis is not required for male pronuclear formation in bovine zygotes

Zygote ◽  
1996 ◽  
Vol 4 (1) ◽  
pp. 41-48 ◽  
Author(s):  
R.C. Chian ◽  
M.A. Sirard
Keyword(s):  
Protein Synthesis ◽  
Calcium Ionophore ◽  
Sperm Nucleus ◽  
Calcium Ionophore A23187 ◽  
Protein Synthesis Inhibitor ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Synthesis Inhibitor ◽  
Parthenogenetic Activation ◽  
Pronuclear Formation

SummaryFollowing fertilisation, the sperm triggers a series of intracellular changes which initiate oocyte activation and pronuclear formation. Oocyte activation can also be induced artificially by several chemicals, such as the calcium ionophore A23187. The sperm nucleus is transformed into the male pronucleus through the interaction of oocyte cytoplasmic factors. The profile of protein synthesis is different in bovine oocytes following fertilisation and parthenogenetic activation. The formation of male and female pronuclei was not blocked by the presence of the protein synthesis inhibitor cycloheximide. These results suggest that bovine oocyte activation by sperm and parthenogenetic activation induce different cytoplasmic responses for protein synthesis and that new protein synthesis is not required for male pronuclear formation in bovine zygotes.

Induced parthenogenetic activation of oocytes of the marsupial Sminthopsis macroura

2004 ◽  
Vol 16 (6) ◽  
pp. 599 ◽  
Author(s):  
Marek Maleszewski ◽  
Lynne Selwood
Keyword(s):  
Polar Body ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Synthesis Inhibitor ◽  
Ionophore A23187 ◽  
Synthesis Inhibitor ◽  
Parthenogenetic Activation ◽  
First Polar Body ◽  
Sminthopsis Macroura ◽  
Second Polar Body

Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmental events, has a low success rate and results from the artificial activation of oocytes, which may not include nuclear maturation. In Sminthopsis macroura, 24-h culture of advanced antral follicles in medium containing 10 μg mL−1 porcine pituitary luteinising hormone (LH) yielded 60% of mature polarised oocytes with the first polar body; follicles cultured in medium without LH yielded only immature oocytes. Parthenogenetic activation of follicular, oviducal or uterine oocytes occurred when a two-step protocol was used. Sixty-one oocytes, exposed to 10 μm calcium ionophore A23187 for 10 min followed by 10 μg mL−1 cycloheximide (protein synthesis inhibitor) for 5 h and then cultured for 20–24 h, were scored for signs of activation, namely extrusion of the second polar body and formation of the pronucleus. In each of 43 oocytes (70%), the extruded second polar body was present. Sixteen oocytes were analysed on slides after fixation and staining and, in 13 oocytes (81%) in this group, the female pronucleus was visible. No activation occurred following incubation of oocytes in medium containing Sr2+ for 5 h (n = 14), 8% ethyl alcohol solution for 8 or 12 min (n = 13) or 10 μm calcium ionophore A23187 (n = 13) for 10–20 min followed by culture for 20–24 h.

A combination of calcium ionophore and puromycin effectively produces human parthenogenones with one haploid pronucleus

Zygote ◽  
2001 ◽  
Vol 9 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Koji Nakagawa ◽  
Shuji Yamano ◽  
Hisayo Nakasaka ◽  
Kenji Hinokio ◽  
Midori Yoshizawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Control Group ◽  
Ionophore A23187 ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Second Polar Body ◽  
Activation Rates ◽  
Pronuclear Formation

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.

Metaphase arrest in newly matured or microtubule-depleted mouse eggs after calcium stimulation

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Ruth M. Moses ◽  
Yoshio Masui
Keyword(s):  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Synthesis Inhibitor ◽  
Ionophore A23187 ◽  
Synthesis Inhibitor ◽  
Microtubule Inhibitor ◽  
Phosphorylated Proteins ◽  
Calcium Stimulation ◽  
Mouse Eggs

SummaryIn mouse eggs arrested at meiotic metaphase II, the increase in intracellular calcium that results from fertilisation induces nuclear formation in both newly ovulated and older eggs. In contrast, the calcium increase that results from exposure to the calcium ionophore A23187 induces nuclear formation in older, but not young, newly ovulated eggs. When treated with the microtubule inhibitor colcemid, and fertilised, young eggs remained at metaphase, but many older eggs formed nuclei, although older eggs treated with colcemid and A23187 remained at metaphase. However, young A23187-treated eggs, young colcemid-treated fertilised eggs, and older colcemid- and A23187-treated eggs, formed nuclei when treated, in addition, with the protein synthesis inhibitor cycloheximide, or the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP). The possibility is discussed that metaphase in newly matured eggs and microtubule-depleted eggs may be maintained by similar mechanisms involving short-lived phosphorylated proteins.

Intracellular pH increase accompanies parthenogenetic activation of porcine, bovine and murine oocytes

2000 ◽  
Vol 12 (4) ◽  
pp. 201 ◽  
Author(s):  
Nancy T. Ruddock ◽  
Zoltán Macháty ◽  
Randall S. Prather
Keyword(s):  
Intracellular Ph ◽  
Prolonged Exposure ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Sea Urchin Egg ◽  
Porcine Oocyte ◽  
Bovine Oocytes

Although an intracellular pH (pH i ) increase at the time of fertilization is necessary for activation of the sea urchin egg, recent reports in the mouse and rat have indicated that there is not a pHi increase during fertilization or during 7% ethanol activation in the mouse. It has been suggested that mammals may have lost the need for a pH i increase at the time of fertilization and the present study reports significant pH i changes during parthenogenetic activation of porcine IVM oocytes, as well as pH i responses to activation in bovine and murine oocytes. Transient intracellular pH changes were found during porcine oocyte activation when using 7% ethanol and with 50 or 100 M calcium ionophore (A23187). Treatment with 200 M thimerosal resulted in an increase in pH i after a delay of approximately 12 min. Murine oocytes showed a significant increase during activation with 7% ethanol and A23187 as well as during prolonged exposure to thimerosal. Bovine oocytes exhibited an increase in pH i only when activated with 50 or 100 M A23187. The final set of experiments aimed to determine whether the porcine oocyte has mechanisms to alleviate induced acidic and alkaline challenges. Both acidic (~20 mM acetic acid) and alkaline (~30 mM ammonium chloride) challenges caused significant changes in pH i that porcine IVM oocytes were capable of recovering from within 35 min. Future studies will focus on determining which of the mechanisms is producing the pH i increase at the time of parthenogenetic activation in the porcine oocyte.

Cell cycle progression of parthenogenetically activated mouse oocytes to interphase is dependent on the level of internal calcium

1992 ◽  
Vol 103 (2) ◽  
pp. 389-396 ◽  
Author(s):  
C. Vincent ◽  
T.R. Cheek ◽  
M.H. Johnson
Keyword(s):  
Cell Cycle Progression ◽  
Polar Body ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Mouse Oocyte ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes

Nuclear maturation of the mouse oocyte becomes arrested in metaphase of the second meiotic division (MII). Fertilization or parthenogenetic activation induces meiotic completion, chromosomal decondensation and formation of a pronucleus. This completion of meiosis is probably triggered by a transient increase in cytosolic calcium ions. When activated just after ovulation by a low concentration of the calcium ionophore A23187, the majority of the mouse oocytes go through a metaphase to anaphase transition and extrude their second polar body but they do not proceed into interphase; instead their chromatids remain condensed and a microtubular metaphase spindle reforms (metaphase III). However, a high percentage of these oocytes will undergo a true parthenogenetic activation assessed by the formation of a pronucleus, when exposed to a higher concentration of the calcium ionophore. The capacity of the mouse oocyte to pass into metaphase III is lost with increasing time post-ovulation. Direct measurement of intracellular calcium with Fura-2 reveals higher levels of cytosolic calcium in aged oocytes and/or using higher concentrations of calcium ionophore for activation. It is concluded that the internal free calcium level determines the transition to interphase.

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