25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets.

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization.

Sonoelastography of Normal Canine Common Calcaneal Tendon: Preliminary Results.

Shear wave elastography (SWE) is a feasible and newly developed ultrasonographic technique which is able to assess elasticity of tissues. The aim of this work was to assess the feasibility of SWE on the normal canine common calcaneal tendon (CCT) evaluating the intra-operator repeatability and reproducibility of single measurements and stiffness of different anatomic CCT portions was examined. Tendons were first evaluated with B-mode ultrasound with a linear probe 8.5 to 10 MHz in longitudinal section with slight flexed tarsocrural joint and a gel-pad. Common calcaneal tendon was divided into three different anatomical regions. Shear wave elastography was performed in each region by two operators and quantitative evaluation (m/s and kPa) was performed on the most representative images. Region of interest (0.15 cm) was settled. Intraclass correlation coefficient (ICC) results were classified using the following scale: 0.00 to 0.20 = poor; 0.20 to 0.40 = fair; 0.40 to 075 = good; >0.75 = excellent. Ten adult dogs were enrolled. Intra-operator ICC values were >0.75 for both operators in every tendon portion. Inter-operator SWE ICC values for m/s measurements were 0.3, 0.61 and 0.61 for the enthesis, intermediate portion and the myotendinous junction respectively; for kPa measurements, values were respectively 0.3, 0.7 and 0.81. The three CCT portions were significantly different in stiffness (p-value < 0.001 for both m/s and kPa measurements). These preliminary results provide evidence that SWE is potentially appliable to assess mechanical properties of canine CCT affected by tendinopathies.

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.

A short exposure to polychlorinated biphenyls deregulates cellular autophagy in mammalian blastocyst in vitro.

BACKGROUND: Polychlorinated biphenyls (PCBs) are common environmental contaminants that represent an important risk factor of reproductive disorders in chronically exposed human populations. However, it is not known whether a short accidental exposure of embryos to PCBs before implantation might influence their further development and whether the effect might be reversible. METHODS AND RESULTS: To this aim, in vitro-matured sheep blastocysts were incubated with 2 or 4 µg/ml Aroclor 1254 (A1254), a mixture of 60 PCB congeners for 48 h after which blastocyst proliferation and ability for outgrowth in vitro were assessed. Blastocysts exposed to A1254 showed: (i) reduced proliferation and cell number (particularly in the inner cell mass compartment); (ii) accumulation of vacuoles and lipid droplets, diffused mitochondrial damage and up-regulation of autophagy markers (ATG6 and LC3), all signs indicative of deregulated autophagy, and (iii) massive cell death. Although exposed embryos resumed growth following A1254 removal, their subsequent development remained severely perturbed. CONCLUSIONS: These findings indicate that short exposure of blastocysts to PCBs leads to its damage characterized by deregulated autophagy and subsequent cell death.

Efficient production and cellular characterization of sheep androgenetic embryos.

The production of androgenetic embryos in large animals is a complex procedure. Androgenetic embryos have been produced so far only in cattle and sheep using pronuclear transfer (PT) between zygotes derived from in vitro fertilization (IVF) of previously enucleated oocytes. PT is required due to the poor developmental potential of androgenotes derived from IVF of enucleated oocytes. Here we compare the developemt to blastocyst of androgenetic embryos produced by the standard pronuclear transfer and by fertilization of oocytes enucleated in Ca2+/Mg2+-free medium, without pronuclear transfer. The enucleation in Ca2+/Mg2+-free medium abolished almost completely the manipulation-induced activation, significantly improving the development to blastocyst of the androgenetic embryos (IVF followed by PT; 18.6%: IVF only; 17.7%, respectively). Karyotype analysis of IVF revealed a similar proportion of diploid embryos in androgenetic and control blastocysts (35% and 36%, respectively), although mixoploid blastocysts were frequently observed in both groups (64%). Androgenotes had lower total cell numbers than control and parthenogenetic embryos, but more cells in ICM cells comparing to parthenogenotes (30.42 vs. 17.15%). Higher expression of the pluripotency-associated gene NANOG, and trophoblastic-specific gene CDX2, were also observed in androgenotes compared to parthenogenotes and controls. The global methytion profile of androgenetic embryos was comparable to controls, but was lower than parthenogenetic embryos. The cell composition and methylation pattern we have detected in monoparental sheep monoparental embryos are unprecedented, and differ considerably from the standard reference mouse embryos. Altogether, these finding indicate significant differences across species in the molecular mechanisms regulating early development of monoparental embryos, and highlights the need to study postimplantation development of androgenetic embryos in sheep.

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

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly 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 developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.

Developmental and functional evidence of nuclear immaturity in prepubertal oocytes.

BACKGROUND: The nuclear compartment has been proposed as responsible for the developmental arrest of prepubertal mouse oocytes while the studies on prepubertal sheep and cow oocyte model suggested the cytoplasm immaturity accounts for this failure. METHODS: The apparent disagreement on the causes of developmental defects between these two species prompted us to study: (i) follicular and oocyte growth allometry in lambs, (ii) oocyte compartment (nucleus versus cytoplasm) responsible for developmental failure by nucleus exchange between lamb and adult sheep oocytes, (iii) nucleolar features of prepubertal oocytes by ultrastructural observation and (iv) in vivo developmental survey of prepubertally derived embryos. RESULTS: The oocyte growth inside the follicle is asynchronous during prepuberty. The nuclear transfer revealed that the lamb nucleus was responsible for developmental failure. Immature fibrillogranular structure of the nucleolus has been revealed in small lamb oocytes and also in a few adult-size lamb oocytes. Studies in vivo revealed a high occurrence of developmental arrest of prepubertal derived fetuses, which we have attributed to the low genome-wide methylation detected in prepubertal oocytes. CONCLUSIONS: Our studies have indicated incomplete nuclear maturation of prepubertal gamete. The implication of this finding suggests caution when the strategy of rescue of prepubertal oocytes for assisted fertilization is considered such as in the case of therapeutic treatment which precludes the maintenance of fertility of sexually immature patients.