Sheep: the first large animal model in nuclear transfer research.

The scope of this article is not to provide an exhaustive review of nuclear transfer research, because many authoritative reviews exist on the biological issues related to somatic and embryonic cell nuclear transfer. We shall instead provide an overview on the work done specifically on sheep and the value of this work on the greater nuclear transfer landscape

Reducing the time of spermatozoa/oocyte interaction improves embryonic development in sheep.

In vitro fertilization (IVF) is a well-established Assisted Reproductive Technique (ART) routinely applied in human infertility treatment and in large animals, both to boost reproductive performances of selected genotypes, and also for basic research. Despite its value, IVF has seen very little progress in the last two decades, and relies to established paradigms, like overnight sperm-egg co-incubation. However, the long exposure to the relatively high spermatozoa concentration in a dish increases the risk of polyspermy and could be detrimental for early embryonic development. In our work we have identified the time window within which the fertilization occurs in order to refine the procedure and reduce sperm-egg co-incubation, comparing embryo development after short (sIVF) and overnight spermatozoa/oocyte co-incubation (o/nIVF). A total of 144 in vitro matured sheep oocytes were co-incubated with spermatozoa in IVF medium [synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16µM isoproterenol]. Then, small batches of oocytes were collected every 30 minutes to check for the presence of a fertilizing spermatozoon. To assess that, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection respectively. Pronuclear formation (PN) and embryo development were evaluated after 16 hours (PN), 24 hours (2 cells) and 7 days of culture (blastocyst). We found that spermatozoa engaged with the ZP not earlier than 90 minutes and penetrated the oocytes within 3-4 hours after IVF. A trend for a lower polyspermic fertilization (>2PN) was detected in sIVF (7.7%) vs o/nIVF (18.4%,). Likewise, cleavage and blastocyst rate were higher after sIVF compared to o/n-IVF (2-cells: 38.6% vs 23.8%; blastocyst: 18.1% vs 9.5%; sIVF vs o/nIVF). This work demonstrates that 4 hours spermatozoa/oocyte interaction are sufficient to achieve fertilization, reduce polyspermy and improve early embryonic development in sheep

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes.

ABSTRACTAndrogenetic embryos are useful model for investigating the contribution of the paternal genometo embryonic development. Little work has been done with androgenetic embryo productionin domestic animals. The aim of this study was the production of diploid androgeneticsheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro;parthenogenetic and normally fertilized embryos were also produced as a control. Fifteenhours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored forthe number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid,and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportionof oocytes with polyspermic fertilization increased linearly with increasing spermconcentration. After IVF, there was no significant difference in early cleavage and morula formationrates between the groups, while there was a significant difference on blastocyst developmentbetween IVF, parthenogenetic, and androgenetic embryos, the last ones displayingpoor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploidandrogenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgeneticembryonic development, we produced diploid androgenetic embryos through pronucleartransfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploidembryos and transferred into the perivitelline space of other haploid embryos, and the zygoteswere reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfersignificantly increased blastocyst development (IVF, parthenogenetic, androgenetic:Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly,the Haploid Diploid group showed the highest development to blastocyst stage.The main findings of our study are: (1) sheep androgenetic embryos display poor developmentalability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryosproduced by pronuclear exchange developed in higher proportion to blastocyst stage,particularly in the Diploid–Haploid group. In conclusion, pronuclear transfer is an effectivemethod to produce sheep androgenetic blastocysts.[...

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to mammalian somatic cells

AbstractLate Embryogenesis Abundant (LEA) proteins are commonly found in organisms capable of undergoing reversible dehydration - “anhydrobiosis”. Here, we have produced three LEA proteins: pTag-RAB17-GFP-N,Zea maysdehydrin-1dhn, expressed in the nucleo-cytoplasm; pTag-WCOR410-RFP,Tricum aestivumcold acclimation proteinWCOR410, binding to cellular membranes, and pTag-LEA-BFP,Artemia franciscanaLEA protein group 3 that targets the mitochondria. Somatic cells transfected with three LEA proteins were subjected to desiccation under controlled conditions, followed by rehydration, viability assessment and membrane/mitochondria functional tests were performed. Results shown that LEA protect cells from desiccation injury. Cells expressed all LEA proteins shown very high percentage of viable cells (58%) after four hour of desiccation compare to un-transfected cells (1% cell alive). Plasmalemma, cytoskeleton and mitochondria appeared unaffected in LEA-expressing cells, confirming their protective action during the entire desiccation and rehydration process. Here, we show that natural xeroprotectants (LEA proteins) transiently expressed in somatic cells confer them desiccation tolerance.</jats:p

Sviluppo post-impianto di embrioni partenogenetici ovini

Tlie objective of this work is tlie assessiiient of tlie post-iinplantatioiidcvelopnieiit of sheep pai-tlieiiogeiietic eiiibryos afier traiisfer iiito previoiisly syncliroiiizedcwcs 7 days iilier oestriis. Iii Vitio Activated (IVA: 11. 337) aiid Iii Vitro Fcrtilizctl (IVF. il.108) coiitrol eiiibryos werc siirgically transferred iiito previoiisly syiiclironized ewes. 130thgroiips of eiiibryos were surgically recovered at da). 22iid of gestatioii. A groiip of teil ewes wasiiatiirally inated. and foetiises recovered also at day 32iid as a liirtlier coiitrol. Post-iiiiplantationviability was reduced iii IVF aiid IVA Iòetiises, coiiipariiig to coiitrol ones ( 10.1%, 8.8% aiid85% respectively). Foetal growtli was also liiiearly reduccd (coiitrol fbetiises 0.49 ciii: IVF. 0.39ciii: IVA, 0.36 ciii). coiifiiiiiiiig previoiis data. Fiirtlier stiidies are curreiitly iii progress iii oiirlaboratoi-y to uiiderstaiid tlie reasoiis for siicli developiiieiital failures. focusiiig oii placenta1aiigiogeiiesis diiring the peri-iinplaiitatioii pliase.[...