Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

Biotechnological bases of the development of cloned pig embryos

The term ‘clone’ in animal biotechnology refers to an organism derived from non-sexual reproduction, which is both a direct offspring and a genetic copy of the parent organism. To date, the pig appears to be the most interesting object in cloning research. Somatic cell nuclear transfer in pigs has a wide range of potential applications in various fields of human scientific and economic activities. However, the efficiency of producing cloned embryos in swine is still lower than that of other livestock species, in particular horses and cattle. Somatic cell nuclear transfer is a technically complex multi-stage technology, at each stage of which the pig oocytes, which are more susceptible to changes of surrounding conditions, are affected by various factors (mechanical, physical, chemical). At the stage of oocyte maturation, changes in the cell ultrastructures of the ooplasm occur, which play an important role in the subsequent nuclear reprogramming of the transferred donor cell. Before transfer to the oocyte donor somatic cells are synchronized in the G0/G1 stage of the cell cycle to ensure the normal ploidy of the cloned embryo. When removing the nucleus of pig oocytes maturated in vitro, it is necessary to pay attention to the problem of preserving the viability of cells, which were devoid of their own nuclear material. To perform the reconstruction, a somatic cell is placed, using micro-tools, in the perivitelline space, where the first polar body was previously located, or in the cytoplasm of an enucleated oocyte. The method of manual cloning involves the removal of the oocyte nucleus with subsequent fusion with the donor cell without the use of micromanipulation techniques. The increased sensitivity of oocytes to the environmental conditions causes special requirements for the choice of the system for in vitro culture of cloned pig embryos. In this work, we have reviewed the modern methods used for the production of cloned embryos and identified the technological issues that prevent improving the efficiency of somatic cloning of pigs.