Cobalamin supplementation during in vitro maturation improves developmental competence of sheep oocytes

Abstract Pregnancies obtained by Assisted Reproductive Technologies are at higher risk of miscarriage than those obtained naturally. Previously, we reported impaired placental vascular development of in vitro produced (IVP) sheep embryos and defective DNA methylation in the placentae of those embryos. One reason behind these observed defects may be an impaired One Carbon Metabolism (OCM) The present study was performed to test the hypothesis that Cobalamin (Vitamin B12, an important OCM co-factor) supplementation during IVM corrects DNA methylation of IVP embryos and, consequently, ameliorates placental vasculogenesis. To this aim, embryos derived from oocytes matured with Cobalamin (B12 group) or without (negative control group, −CTR) were transferred to synchronized recipient sheep. At day 20 of pregnancy, collected embryos were morphologically evaluated while placentae were subjected to qPCR and histological analysis. The positive control group (+CTR) consisted of conceptuses obtained from naturally mated sheep. Results showed an increased fertilization rate in the B12 group vs –CTR (69.56% vs 57.91% respectively, P = 0.006) not associated with quantitative improvement in blastocyst and/or implantation rate (44.32% vs 36.67% respectively, P > 0.05). Moreover, Cobalamin supplementation during oocyte IVM ameliorated resulting conceptuses quality, in terms of placental vascularization (vessels' maturity and vasculogenetic factors' expression). The expression of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) was also improved in placentae from the B12 group. In conclusion, Cobalamin supplementation during oocyte IVM improves IVP embryo quality. These results suggest that Cobalamin should be included in standard IVM media

Comparison of slow and rapid freezing for long term storage of freeze-dry ram spermatozoa

Semen lyophilization is an interesting technique that might be a cheap alternative to long-term storage under liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 [1] demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. From this work on, the most used approach for lyophilisation is that of deep-freezing, that is directly immersing the semen sample into liquid nitrogen before vacuum drying. Recently we have shown that it is possible to establish a "dry" bank of ejaculated and epidydimal freeze-dried ram spermatozoa [2, 3]. In order to improve and make the technique more reliable, here we focused on the freezing phase, comparing two different protocols: i) Fast-freezing, where the semen is plunged directly into liquid nitrogen (LN group); ii) Slow-freezing, where the sample is progressively cooled to a final temperature of -50°C (SL group). Briefly, for the preparation of the LN group sample the protocol reported in [2] was followed, while for the SL group the semen was frozen with a freezing rate of 1°C/min until -50°C degrees, when the sample was placed inside the lyophilizer. Dry spermatozoa from both groups was used for Intracytoplasmic Sperm Injection (ICSI) and the embryo development was evaluated at 24h (2-Cells stage) and 7 days (expanded blastocyst) after fertilization. At 24h post fertilization the SL-group showed a higher number of cleaved embryos than LN-group (42/100 (42%) versus 19/75 (25.3%), P=0.0253, SL and LN respectively). At 7 days after fertilization the blastocyst rate in SL-group was higher (7/100 (7%)) than in LN-group (2/75 (2.7%)), although not statistically different. Our data shows that lyophilisation can be conveniently achieved in ram spermatozoa without previous freezing in liquid nitrogen, thus simplifying the procedure. This data supports the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen

Alternative strategies for nuclear reprogramming in somatic cell nuclear transfer (SCNT)

Twenty years passed by since the production of Dolly the sheep, but despite significant technical progress has been achieved in the manipulation procedures, the proportion of offspring following transfer of SCNT embryos has remained almost unchanged in farm animals. Remarkable progress has been obtained instead in laboratory animals, particularly by Japanese Groups, in the mouse. However, the nuclear reprogramming strategies tested in mouse do not always work in farm animals, and others are difficult to be implemented, for require complicated molecular biology tools unavailable yet in large animals. In this review we put in contest the previous work done in farm and laboratory animals with recent achievements obtained in our laboratory, and we also indicate a road map to increase the reliability of SCNT procedures

Ultrastructural analysis reveals abnormal mitochondria in cloned blastocysts

Somatic cell nuclear transfer (SCNT) is a powerful technique, but still very inefficient despite 20 years passed by since the cloned mammal was born. We have recently shown that the major cause of abnormalities observed in cloned fetuses are mitochondrial dysfunctions in placenta collected from cloned sheep. Investigations on mitochondria in SCNT are limited to the mtDNA hetero/homoplasmy in cloned offspring, whereas no data is available for an eventual role of mitochondria dysfunction on the developmental failure of cloned animals. Here we wanted to know whether mitochondrial abnormalities are observed already in cloned blastocysts since mitochondrial replication does not occur after the hatched blastocysts stage. SCNT and in vitro processed (IVP) blastocysts were produced and analysed for mitochondrial structure and functionality. First, embryos were analysed using transmission electron microscope (TEM). Drastic differences in mitochondrial structure between SCNT and IVP blastocysts were observed. Decrease density of mature mitochondria, very high degree of cytoplasmic vacuolisation, numerous cytoplasmic vesicle and autophagosomes were observed in SCNT blastocysts. Moreover, statistically lower expression of major mitochondrial, autophagic and apoptotic proteins were observed in SCNT embryos. Obtained results clearly shown that mitochondrial abnormalities are already observed in blastocysts stage embryos. It is important to point out that activity of mitochondria are strictly control by nuclear signals, thus, obtained results may suggest that incomplete nuclear reprogramming in cloned nucleus might be responsible also for the impaired mitochondrial function in cloned embryos/fetuses

Developmental peculiarities in placentae of ovine uniparental conceptuses

Genomic imprinting is an epigenetic phenomenon regulating mono-allelic expression of genes depending on their parental origin. Defective genomic imprinting is involved in several placental disorders, such as intrauterine growth restriction and pre-eclampsia. Uniparental embryos, having maternal-only or paternal-only genomes (parthenogenotes [PAR] and androgenotes [AND], respectively), are useful models to study placentation. The aim of this work was to reveal the effect of parental genome (maternal and paternal) on placentation. To do this, uniparental (AND and PAR) and biparental (CTR) in vitro produced sheep embryos transferred to recipient females were collected at day 20 of pregnancy and their placentae were analyzed. qPCR analysis showed that imprinted genes (H19, IGF2R and DLK1) were expressed accordingly to their parental origin while the expression f DNA methyltransferases () was disregulated, especially in PAR (P < 0.05). AND placentae were significantly hypomethylated compared to both PAR and CTR (P = 0.023). Chorion-allantoid of AND showed impaired development of vessels and reduced mRNA expression of vasculogenetic factors (ANG2 P = 0.05; VEGFR2 P< 0.001; TIE2 P < 0.001). Morphologically, PAR placentae were characterized by abnormal structure of the trophoectodermal epithelium and reduced total number (P<0.03) of Trophoblastic Binucleate Cells. A reduced implantation rate of both classes of uniparental embryos (P<0.03) was also noted. Our results provide new insights into the characterization of uniparental embryos and demonstrate the complementary role of parental genomes for the correct establishment of pregnancy. Thus, our findings may suggest new targets to improve our understanding of the origin of imprinting-related placental dysfunction

Evidence of placental autophagy during early pregnancy after transfer of in vitro produced (IVP) sheep embryos

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos