Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice

BACKGROUD: Transferring a germinal vesicle (GV) from an aged woman's oocyte into ooplasm from a younger woman has been proposed as a possible way to overcome the problem of age-related decline in female fertility. Here we assessed this possibility by determining whether ooplasts derived from young mice could rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. METHODS: Three groups of reconstructed oocytes, young GV-young cytoplast (group YY), aged GV-young cytoplast (group AY), and young GV-aged cytoplast (group YA), were created by micromanipulation and electrofusion. RESULTS: Nuclear transplantation was successful in 89.8-94.4% of GV-ooplast complexes, and maturation rate of the reconstructed oocytes was 93.5-97.9%. Confocal microscopy analysis showed a significantly higher rate (49.2%) of chromosome misalignment in ageing mice than in young mice (16.9%), and 57.1% of oocytes in group AY exhibited chromosome misalignment, while the abnormality rate in groups YY and YA was 16.3 and 16.7% respectively. Calcium imaging showed that the three groups of reconstructed oocytes exhibited a similar pattern of calcium oscillations upon stimulation with bovine sperm extracts. Fertilization rate and developmental capacity to 2-cell embryos were also similar among the three groups of oocytes. CONCLUSIONS: Our findings suggest that: (i) the ooplasm from young mice could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice; and (ii) behaviour of chromosome alignment over metaphase spindle is predominantly determined by GV material.     

Effects of myo-inositol on the in-vitro maturation and subsequent development of mouse oocytes

BACKGROUND: The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro. METHODS AND RESULTS: We performed a controlled prospective study using female ICR strain mice superovulated with pregnant mare's serum gonadotrophins. Cumulus-enclosed germinal vesicle (GV) oocytes were randomly cultured in medium with or without MI supplementation. The kinetics of GV breakdown after 4 h of incubation was significantly higher in oocytes incubated with 30 mmol/l of MI than in controls (P < 0.001). Accordingly, this concentration of MI was used for subsequent experiments. The proportion of metaphase II oocytes achieved after 24 h of culture, their fertilization and cleavage rates were significantly higher in the MI-treated group (P < 0.01, P < 0.05, P < 0.05 respectively). This group also demonstrated significant improvement in postimplantation development after transferring the 2-cell embryos to pseudopregnant mice. Confocal microscopy revealed spontaneous intracellular Ca(2+) oscillations within competent GV oocytes and treatment with MI caused an earlier onset of these Ca(2+) signals. CONCLUSIONS: Our results suggest that MI may affect meiotic progression of mouse GV oocytes possibly by enhancing the intracellular Ca(2+) oscillations. Supplementation of MI in culture medium may be useful for human oocyte maturation.     

Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3

BACKGROUND: Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. METHODS: Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. RESULTS: Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. CONCLUSIONS: Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.     

Fertilization and early embryology: Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals

The objective of this investigation was to determine whetherintracytoplasmic sperm injection (ICSI) can be performed inthe mouse. Metaphase II oocytes were obtained from F1 hybridmice (C57BLx CBA) by i.p. injections of 10 IU pregnant mare'sserum gonadotrophin (PMSG) and human chorionic gonadotrophin(HCG) administered 48 h apart. Oocytes with cumulus oophoruswere retrieved 13–14 h post HCG. Cumulus was dispersedwith 0.1% hyaluronidase. Mouse spermatozoa were obtained fromthe cauda epididymides of males of the same strain. The spermatozoawere processed by the standard swim-up procedure. The harvestedspermatozoa were then incubated for 1.5 h to allow capacitation.Healthy oocytes were injected with 3–4 pi 5 mM Ca²⁺, followedby one live morphologically normal spermatozoon into the cytoplasmat intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cellembryos that developed from oocytes injected with Ca²⁺ and spermatozoaranged between 29.5 and 36.5% in all groups, with no statisticaldifference between treatments. Chromosomal analysis showed thattwo-thirds of the ICSI-derived 2-cell embryos were diploid.The proportion of parthenogenetically activated embryos in theICSI groups was similar to that in the control group (8–10%)which was injected with Ca²⁺ and polyvinyl pyrrolidone only.The proportion of blastocysts that developed in culture fromthe ICSI-derived 2-cell embryos was of the order of 36–42%.Some blastocysts were used for cell number counts. There wasa significant increase in total and inner cell mass counts ofblastocysts in which the spermatozoon was injected at 2 and3 h following Ca²⁺. The remaining blastocysts were transferredto day 3 pseudopregnant mice, of which 33% subsequently becamepregnant. Of the blastocysts transferred, 16–25% developedto term in vivo. No deformities were observed in the pups. Webelieve this is the first report of live-birth following mouseICSI.     

Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos

In this study, the use of Ca²⁺/Mg²⁺-free medium for biopsy ofhuman embryos at the 4- to 10-cell stage on the third day ofdevelopment was evaluated. When compared with control mediumcontaining normal concentrations of Ca²⁺ and Mg²⁺ ions, theuse of Ca²⁺/Mg²⁺ -free medium allows an easier removal of blastomeresas illustrated by a lower rate of cell lysis as well as by ashorter time needed to perform the procedure. Subsequent embryodevelopment to the blastocyst stage is not affected by the choiceof biopsy medium, not even when embryos are exposed to the mediumfor 45 min. The use of Ca²⁺/Mg²⁺-free medium thus allows foran easier biopsy procedure during preimplantation genetic diagnosis,while it does not result in a loss of developmental potentialof the embryo to the blastocyst stage.