Bi-directional communication with the cumulus cells is involved in the deficiency of XY oocytes in the components essential for proper second meiotic spindle assembly

Abstract

The oocyte becomes competent for embryonic development by involving mutual communication with cumulus cells (CCs) during folliculogenesis. How this communication takes place under physiological conditions is not fully understood. Current study examined oocyte-CCs communication in the XY sex-revered female mouse. We have previously found that the XY oocyte is defective in its cytoplasm, causing abnormal MII-spindle assembly and a failure in embryonic development. Our present study showed that transcript levels of Pfkp, Pkm2 and Ldh1 involved in glycolysis were lower in the CCs surrounding XY oocytes than in those surrounding XX oocytes. ATP contents in XY oocytes were also lower than those in XX oocytes, suggesting that lower glycolytic gene expression in CCs resulted in lower ATP contents in the enclosed oocyte. Co-culture of oocytectomized CC-oocyte complexes (COCs) with denuded oocytes showed that XY oocytes were less efficient than XX oocytes in promoting glycolytic gene expression in CCs. Furthermore, both glycolytic gene expression levels in CCs and ATP contents in oocytes of XY COCs increased to similar levels to those of XX COCs after culture for 20h in the presence of milrinone (=preincubation), which prevented spontaneous oocyte maturation. By increasing ATP levels in XY oocytes by either COC preincubation or ATP microinjection into oocytes prior to in vitro maturation, an improvement in MII-spindle assembly was observed. We conclude that the XY oocyte produces lesser amounts of paracrine factors that affect its companion CCs, which in turn make the ooplasm deficient in its components, including ATP, essential for MII-spindle assembly.

A lack of coordination between sister-chromatids segregation and cytokinesis in the oocytes of B6.YTIR (XY) sex-reversed female mice

“…We have previously observed that MII-spindles are always positioned in the perpendicular orientation in the IVM-oocytes from XY females 17, 29 . However, as we arbitrarily mounted oocytes on histology slides, we could not exclude the possibility that the oocytes were positioned so that spindles appeared to be perpendicular but were actually parallel to the oolemma.…”

“…We have previously reported that meiotic spindles in the IVM oocytes from XY females are loosely organized and possibly in a wrong orientation 17, 29 . We had speculated that the improper spindle orientation might be responsible for the failure in the subsequent second meiotic division.…”

Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes

“…A recent article showed that the percentages of mature oocytes having a normal spindle and the blastocyst formation rate were significantly lower in oocytes denuded 22 hours than 44 hours after the start of IVM [26]. The metaphase-II spindle assembly has been reported to be improved by elevated intra-ooplasmic ATP levels following either COC preincubation or ATP microinjection into the oocytes before IVM [35]. The current results suggest that disruption of the communication between CCs and the oocyte 20 hours after the start of IVM may have a detrimental effect on the intra-ooplasmic content of ATP.…”

Glycine increases preimplantation development of mouse oocytes following vitrification at the germinal vesicle stage

“…Denuded oocytes were fixed in a solution containing 2% paraformaldehyde and microtubule stabilizing buffer56 for 60 min at room temperature. Then, oocytes were transferred into blocking buffer (PBS supplemented with 1% BSA and 0.01% Triton X-100) for 1 h at room temperature and incubated with mouse anti-alpha-tubulin-FITC monoclonal antibody (Sigma) diluted 1:200 in the blocking buffer for 60 min at room temperature.…”

“…30 oocytes in each group were snap-frozen in a microfuge tube containing 200 μl of ultrapure water and stored at −80 °C. ATP contents in oocytes were determined by using the assay kit (Bioluminescent Somatic Cell Assay Kit, FL-ASC, St Louis, MO) based on the luciferin–luciferase reaction as previously described56. The bioluminescence of each sample was measured by high-sensitivity luminometer (Berthold LB 9508, Germany) including a standard curve with different ATP concentrations in each assay.…”