Nuclear reprogramming: what has been done and potential avenues for improvements

Abstract A major challenge for reproductive biologists is the development of novel strategies to improve cloning efficiency. Even in species for which cloning is relatively successful, like cattle, the efficiency is still unacceptably low. In this review article we critically analyse all approaches that have been suggested by different laboratories in the field so far. As will be discussed below, so far none of these gives rise to a dramatic increase in cloning efficiency. Possibly, a multi-step approach including a pre-treatment of donor cells to modify their chromatin, along with improved culture system for cloned embryos would be the most promising

Whole genome integrity and enhanced developmental potential in ram freeze‑dried spermatozoa at mild sub‑zero temperature

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to − 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen

Freeze-Dried Somatic Cells Direct Embryonic Development after Nuclear Transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies

Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost

Developmental and functional evidence of nuclear immaturity in prepubertal sheep oocytes.

ABSTRACT- Prepubertal sheep embryo transferred to foster mother for development to term is able to progress through the first part of gestation, while further fetal development is often interrupted. Disruption of maternal primary imprinting has been proposed as responsible for similar developmental arrest of neonatal mouse oocytes. On the other hand, in ovine and bovine species several structural and functional deficiencies of the cytoplasm are widely reported as causes of low developmental potential of prepubertal oocyte. In the light of these observations, developmental competence of each compartment of lamb oocyte, nucleus and cytoplasm, were studied first. To identify the compartment responsible for developmental failure, nucleus exchange between lamb and adult sheep oocyte followed by sperm injection into the reconstructed egg, were performed. Using this approach, we revealed that when lamb oocytes smaller than those from adult sheep were used for nuclear transfer, development of oocytes reconstructed with either sheep or lamb nucleus was arrested before blastocyst stage. This demonstrates that smaller oocytes lack both cytoplasmic and nuclear competence. Surprisingly, developmental progress of eggs reconstructed from nuclear exchange between adult and lamb oocytes similar in diameter, was significantly different for sheep- and lamb-nucleus derived embryos (18% vs. 0% blastocysts, respectively), indicating oocytes with diameters similar to adult still incompetent in terms of nuclear preparation. Secondly, to reveal if the epigenotype of prepubertal oocyte is appropriate for supporting normal development, global methylation level was assessed using anti-5-methylcytidine antibody. High levels of antibody incorporation were observed in nuclei of adult sheep oocytes (n=58) confirming fully established genome methylation while the intensity of nuclear immunofluorescence was invariably weak in similar size lamb oocytes (n=65) or even absent in more than half of smaller oocytes (34/62), indicative of epigenetic immaturity. In this study, we present developmental and functional outlines of nucleus immaturity reinforced by previous reports of late pregnancy loss following transfer of lamb embryos, leading to further insight on the causes of prepubertal oocytes developmental defects.[...