Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level

Abstract

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.

Effect of in vitro and in vivo conditions on development of parthenogenetic rabbit embryos after vitrification

“…11" " similar to previous ones in our laboratory [27,41]. Our results are lower than those 229" obtained in the pig, which showed about 70% of live embryos in vitro after vitrification 230" [8,22,23].…”

Embryo vitrification in rabbits: Consequences for progeny growth

“…To this end, rabbit embryo banking has allowed the preservation and spread of genetically superior animals over different countries [34] and the possibility of evaluating the genetic gain in maternal lines [4][5][6][7] without cumulative genetic drift variance [35]. Nevertheless, it is known that these techniques induce changes in environ-mental and maternal side effects modifying the embryo gene expression and methylation pattern [19,20,36], the transcriptomic and proteomic placental profile [21], and their viability [29,37], but little is known regarding the long-term effects on the derived progeny. Some authors postulate that these procedures do not induce major anomalies but can lead to morphologic and behavioral features in adult mice derived from frozen embryos [16].…”

“…Nevertheless, it must be pointed out that a recent study has reported that embryo cryopreservation and transfer pro-cedures induce changes in the placental function, altering the lipolytic-lipogenic pathway [21], which in fact could affect future organ development, as reported in a series of studies in swine [43,44]. In addition, several authors re-ported modifications in adult organs size due to embryo manipulation.…”

“…Over the past decade, research works have questioned whether embryo cryopreservation and transfer procedures are neutral to survivors [16][17][18], as preimplantation em-bryos are removed from their natural environment and subjected to manipulation (cryopreservation and transfer procedures) which in fact affect their RNA expression [19,20], placental transcriptome, and proteome [21].…”

Embryo Manipulation Techniques in the Rabbit

“…So, asynchrony between vitrified embryos and recipient females is applied to obtain higher rates of embryos developed to term [73]. Cryopreservation and transfer procedures affect the RNA expression [74], and consequently the placental transcriptome and proteome [75]. Vitrification also affects foetal survival and growth and modifies the placental development at term [60,76] and growth from birth to adult age [77].…”