Ultrastructural modifications in bovine oocytes maintained in meiotic arrest in vitro using roscovitine or butyrolactone

Abstract

Butyrolactone-I (BL-I) and roscovitine (ROSC) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes for 24 hr without having a negative affect on subsequent development to the blastocyst stage. The aim of the present study was to describe the morphological changes occurring in fully grown immature and in vitro matured bovine oocytes following exposure to either BL-I or ROSC for 24 hr at concentrations known to be consistent with normal development. Immature bovine cumulus oocyte complexes, recovered from the ovaries of slaughtered heifers, were incubated for 24 hr in the presence of one of the inhibitors. They were then either fixed immediately and processed for transmission electron microscopy (TEM), or cultured for a further 24 hr in the absence of the inhibitor, in conditions permissive to maturation, and subsequently processed for TEM. A control group of oocytes were processed for TEM immediately upon recovery (0 hr) or following in vitro maturation (IVM) for 24 hr. In general, incubation with either inhibitor disrupted the integrity of the surrounding cumulus cells and affected their subsequent expansion during IVM. Within the oocyte cytoplasm, swelling of the mitochondrial cristae was immediately noticeable following meiotic inhibition in the presence of ROSC, while an increased population of pleomorphic mitochondria and mitochondria with electron lucent matrices following BL-I treatment was not observed until after the subsequent IVM period. Both inhibitors caused degeneration of the cortical granules, effectively reducing the population, most noticeably following IVM. At the level of the nucleus, both inhibitory treatments caused convolution of the nuclear membrane, furthermore, aberrant structures were observed within the nucleoplasm of ROSC-treated cumulus oocyte complexes (COCs). In conclusion, while it has been shown that inhibition of meiotic resumption using specific cdk inhibitors is possible and that such oocytes are capable of undergoing maturation, fertilization, and early embryo development, there is as yet no definitive proof that oocytes treated in this way can ultimately give rise to normal offspring. We have shown here that some modifications are induced in the oocytes at the ultrastructural level. Whether or not these modifications are compatible with normal gestation and the birth of a live calf remain to be elucidated.

Retinoid‐dependent mRNA expression and poly‐(A) contents in bovine oocytes meiotically arrested and/or matured in vitro

“…Accumulation of cyclin B1 transcripts were found in bovine oocytes matured in vivo but not in oocytes matured in vitro in the presence of serum (Lonergan et al, 2003a), in contrast to our findings in oocytes matured in chemically defined conditions. Levesque and Sirard (1996) did not find immunodetected cyclin B1 in bovine oocytes until meiotic resumption, but our results are in agreement with authors that detected transcripts for cyclins in immature oocytes (Winston, 2001;Vigneron et al, 2002).…”

“…In addition, after comparing our gene expression profile found for Cyclin B1, MnSOD, and G6PDH with a previous study using a serum-supplemented maturation medium (Lonergan et al, 2003a), it is again demonstrated that serum yields artifacts. In response to RA, the bovine oocyte does not modify expression of cyclins B1 and HI genes, essential regulators of cell cycle, and the retinoid is unlikely to generate oxidative stress.…”

“…However, Lonergan et al (2003a) found similar abundance of both gene transcripts between in vitro matured and immature oocytes from slaughterhouse ovaries. In contrast, oocytes matured in vitro, recovered just before the LH surge, and oocytes matured in vivo, showed decreased MnSOD and increased G6PDH, in agreement with our results.…”

“…In cattle, gene expression of MnSOD (Lequarre et al, 2001;Lonergan et al, 2003a) and G6PDH (Liu et al, 2000) was found in immature and in vitro matured oocytes (Lequarre et al, 2001;Lonergan et al, 2003a). However, Lonergan et al (2003a) found similar abundance of both gene transcripts between in vitro matured and immature oocytes from slaughterhouse ovaries.…”

“…Upon a 9-cis-RA treatment, poly-(A) dramatically increased after meiotic arrest and decreased when these oocytes matured in vitro. The amount of poly-A in the oocyte is associated with a gain in the quality of mRNA stored in the ooplasm (Lonergan et al, 2003a), and defective developmental competence is accompanied by abnormal polyadenylation levels (Brevini et al, 2002).…”