Abstract
Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media. The first sign of meiotic resumption is germinal vesicle breakdown (GVB). Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3. The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h. Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s. The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3. Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations. In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin. Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB. The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation. However, the spontaneous oscillations do not appear to trigger GVB. Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.
Highlights
From the Medical Research Council Experimental Embryology and Teratology Unit, St.George'sHospital Medical School, Cranmer Terrace, London, SW17ORE, United Kingdom
Thfierst sign of meiotic resumptionone is sensitive to InsPs which releases Ca2+into a second is germinal vesicle breakdown (GVB)
The data show that cytosolicCa" oscil- germinal vesicle breakdown (GVB)which in themouse occurs lations apparentlycausedbypolyphosphoinositide within 2 h of release from the follicle.The cellular messengers turnover occur during mammalian oocyte maturationt.hat are responsible for triggering the resumption of meiosis
Summary
Oocyte Recouery-Oocytes were obtained from naturally cycling 68-week-old random bred MF1 mice or 21-24-day-old B6CB (C57B1/ 6JLac X CBA/CaLac) F1 hybrids that weregiven 5IUpregnant mares’ serum gonadotrophin 44-52 h previously. Antral follicles were punctured to release oocytes, and those with adhering granulosa cells were collected. Oocytes were released from antral follicles into M2 (controls), or into M2 supplemented with various drugs as described, and incubated for up to 4 h at 37 “C. Oocytes were incubated for 15 min at 37 “C with 50 PM of the acetoxymethyl (AM) form of the dyes made up in M2 with 0.02% pluronic F-127 [25]. Base-line fluo-3fluorescence from loaded oocytes decreased with time, but since the indo-1 recordings suggested a fairly constant base-line level of intracellular Caz+ the decrease in fluo-3 fluorescence was judged to be due to loss of dye. The diminishing signal was prevented, and a constant level of base-line fluorescence was maintained by the addition of 0.25 p~ fluo-3 AM to thesolution containing the oocytes. Fluo-3 potassium salt (-2 pl of 10 mM fluo-3) and heparin (-2 pl of mg/ml heparin; M, 4000-6000) werepressure-injected [26].Injection of InsP3 was performed as described previously [9]
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