Developmental competence of ovine oocytes after vitrification: differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei

Abstract

Despite many advances in the field of oocyte cryopreservation, the adverse effects of cryopreservation on oocyte competence are still an open challenge in most mammalian species. Using ovine in vitro-matured oocytes, the differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei were systemically investigated to unravel (1) the most critical stage (if any) of oocyte vitrification, (2) the most suitable method (if any) of embryo production for a vitrified oocyte, and (3) differential contributions of male or female pronuclear formation to the poor quality of vitrified oocyte. Although cryoprotectants used during vitrification had some inevitable adverse effects on oocyte competence, the damages caused by low temperature per se (chilling injury) were the main cause of poor quality of vitrified oocytes. When vitrified oocytes underwent either IVF or intracytoplasmic sperm injection (ICSI), embryo development rates were substantially lower than those of fresh ones. In contrast, when vitrified oocytes underwent either parthenogenetic activation (PA) or SCNT, embryo development rates were very similar to those of fresh ones. Evaluation of nuclear morphology after IVF, ICSI, PA, and SCNT oocytes revealed that vitrification had no apparent effect on the female (IVF, ICSI, and PA) and somatic (SCNT) pronuclear formation rates but significantly reduced male pronuclear formation after either IVF or ICSI compared with fresh counterparts. Quantitative analysis of transcripts revealed comparable mRNA abundances of CNX43, HSP90, GMNN, NPM, and OCT4 between vitrified and fresh oocytes, whereas CCNB, ATP1A1, and PAP transcripts were significantly lower in vitrified versus fresh oocytes. Although underlying mechanisms of poor quality of vitrified oocytes are multifactorial, the ability to obtain equivalent development after PA and SCNT, but not IVF and ICSI, in vitrified versus fresh oocytes may argue that the cytoplasm of vitrified oocyte has the necessary components to support in vitro embryonic development of the maternal, even adult somatic cell, chromosomes but fails to do so with sperm chromosomes.

Keywords: IVF; Intracytoplasmic sperm injection; Oocyte; Parthenogenetic activation; SCNT; Vitrification.