Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

Abstract

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.

Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

“…The ladder-like PCR products in CEOs were hardly detectable at 0 h of IVM, while they increased after 12 h of culture and toward the end of IVM (24 h), irrespective of MK addition. The sizes of the ladders observed were, as expected, 185 bp multiples with additional 24 bp oligonucleotides at each DNA end corresponding to the apoptotic internucleosomal fragments (Ikeda et al 2003). When compared at each time point, the apoptotic index was significantly lower in the MK-treatment group at 18 and 24 h of IVM than in the control.…”

“…We previously showed that apoptosis occurs in cumulus cells during IVM of CEOs (Ikeda et al 2003). We tentatively refer to this phenomenon as IVM-induced apoptosis because apoptosis is unlikely to occur in cumulus cells during natural (i.e.…”

“…We tentatively refer to this phenomenon as IVM-induced apoptosis because apoptosis is unlikely to occur in cumulus cells during natural (i.e. in vivo and not artificially stimulated) oocyte maturation (see the discussion in the reference, Ikeda et al 2003). It was also found that apoptosis during IVM was induced irrespective of serum addition, although serum has been shown to protect cultured granulosa cells from apoptosis (Hu et al 2001, Johnson et al 2001.…”

“…However, the concrete action of MK upon cumulus cells remains unknown. Recently, we reported that cumulus cells undergo apoptosis during the IVM period and suggested the negative correlation between the developmental competence of CEOs and the degree of apoptosis of the cumulus cells during IVM (Ikeda et al 2003). On the other hand, MK has been reported to have anti-apoptotic effects on certain types of cells, including neuronal (Owada et al 1999a and1999b) and tumour (Qi et al 2000, Ohuchida et al 2004 cells.…”

“…CEOs were cultured for 0, 6, 12, 18 and 24 h at 39 8C under 5% CO 2 in air. The detection of the nucleosomal DNA ladder of apoptotic cumulus cells was performed using an ApoAlert ligationmediated PCR (LM-PCR) ladder assay kit (Clontech) as reported previously (Ikeda et al 2003). The LM-PCR assay has been reported to be semiquantitative, allowing the comparison of the relative extent of apoptosis in different samples (Staley et al 1997).…”