Daniela Bebbere

The impact of vitrification procedures on in vitro matured (IVM) ovine oocytes mRNA content and ability to undergo successful fertilization, cleavage and embronic development was assessed. Vitrified-warmed (n = 113) and control (n = 140) IVM oocytes were in vitro fertilized and cultured up to blastocyst stage under standard conditions. Vitrified oocytes showed lower cleavage rate (47% vs. 75%, P < 0.001) and development to blastocyst stage (17% vs. 57%, P < 0.001) than controls. In addition, the timings of the first cleavage and blastocysts production were significantly delayed in the vitrified-warmed group (P < 0.001 in both cases). In parallel, we analyzed by reverse transcriptase real-time PCR the relative abundance of beta-actin, H2A.Z histone, Poli A Polimerase (PAP), Heat Shock Protein 90 beta (HSP90 beta), P34(cdc2), Cyclin b, Na/K-ATPase and Type I cadherin (E-Cad) transcripts in single IVM controls (n = 24) and vitrified-warmed oocytes (n = 40). Results were normalized against the exogenous rabbit alpha-globin mRNA standard and the beta-actin housekeeping gene and similarly described a lower abundance of most mRNAs in oocytes subjected to vitrification procedures. When normalized against the exogenous standard mRNA, all transcripts except for beta-actin and H2A.Z showed a significantly different abundance in the two classes of oocytes. The same results were obtained after normalization against the internal standard, except for HSP90 beta and E-Cad transcripts, whose lower abundance in vitrified-warmed oocytes resulted prominent, but not significant (P = 0.083 and P = 0.068, respectively). The oocyte lower transcripts abundance following vitrification might be an early indicator of poor quality in good correlation with the developmental data to blastocyst stage.

Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.

The present study was conducted to investigate the relation between in vitro developmental competence and the expression of a panel of developmentally important genes in germinal vesicle (GV) stage oocytes. One-month-old prepubertal and adult sheep oocytes were used as models of low and high quality gametes, respectively. Cumulus-oocyte complexes (COCs) derived from lambs and ewes were in vitro matured and fertilized, and their cleavage rate at 22, 26, and 32 hr post fertilization and the blastocyst yield were observed to assess their developmental potential. In parallel, the relative abundance (RA) of 11 genes was analyzed by semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay in the two groups of oocytes. We observed similar maturation and fertilization rates in the two groups, but a significant lower rate of cleaved prepubertal oocytes (P < 0.05), a general delay in the timing of their first division (P < 0.01), and a lower blastocysts production (P < 0.05). The analysis of gene expression evidenced no difference in the RA of four transcripts [superoxide dismutase (SOD), ubiquitin, beta-actin, cyclin B] in the two classes of oocytes, but a statistically lower RA of seven messenger RNAs (mRNA) [Na(+)K(+)ATPase, p34(cdc2), Glucose-transporter I (Glut-1), Activin, Zona Occludens Protein 2 (PanZO2), Poli(A)Polymerase (PAP), E-Cadherin (E-Cad)] in the prepubertal oocytes compared to the adult ones. The present data show for the first time in the ovine species that the lower developmental competence is associated with deficiencies in the mRNAs storage during the oocyte growth.

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.