All roads lead to Rome: the many ways to pluripotency.

Cell pluripotency, spatial restriction, and development are spatially and temporally controlled by epigenetic regulatory mechanisms that occur without any permanent loss or alteration of genetic material, but rather through modifications "on top of it." These changes modulate the accessibility to transcription factors, either allowing or repressing their activity, thus shaping cell phenotype. Several studies have demonstrated the possibility to interact with these processes, reactivating silenced genes and inducing a high plasticity state, via an active demethylating effect, driven by ten-eleven translocation (TET) enzymes and an overall decrease of global methylation. In agreement with this, TET activities have been shown to be indispensable for mesenchymal to epithelial transition of somatic cells into iPSCs and for small molecule-driven epigenetic erasure. Beside the epigenetic mechanisms, growing evidences highlight the importance of mechanical forces in supporting cell pluripotency, which is strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, through the activation of specific mechanosensing-related pathways. In this review, we discuss and provide an overview of small molecule ability to modulate cell plasticity and define cell fate through the activation of direct demethylating effects. In addition, we describe the contribution of the Hippo signaling mechanotransduction pathway as one of the mechanisms involved in the maintenance of pluripotency during embryo development and its induction in somatic cells.

Implications of miRNA expression pattern in bovine oocytes and follicular fluids for developmental competence.

Developmental competence determines the oocyte capacity to support initial embryo growth, but the molecular mechanisms underlying this phenomenon are still ill-defined. Changes in microRNA (miRNA) expression pattern have been described during follicular growth in several species. Therefore, aim of this study was to investigate whether miRNA expression pattern in cow oocyte and follicular fluid (FF) is associated with the acquisition of developmental competence. Samples were collected from ovaries with more than, or fewer than, 10 mid-antral follicles (H- and L-ovaries) because previous studies demonstrated that this parameter is a reliable predictor of oocyte competence. After miRNA deep sequencing and bioinformatic data analysis, we identified 58 miRNAs in FF and 6 in the oocyte that were differentially expressed between H- and L-ovaries. Overall, our results indicate that miRNA levels both in FF and in the ooplasm must remain within specific thresholds and that changes in either direction compromising oocyte competence. Some of the miRNAs found in FF (miR-769, miR-1343, miR-450a, miR-204, miR-1271 and miR-451) where already known to regulate follicle growth and their expression pattern indicate that they are also involved in the acquisition of developmental competence. Some miRNAs were differentially expressed in both compartments but with opposite patterns, suggesting that miRNAs do not flow freely between FF and oocyte. Gene Ontology analysis showed that the predicted gene targets of most differentially expressed miRNAs are part of a few signalling pathways. Regulation of maternal mRNA storage and mitochondrial activity seem to be the processes more functionally relevant in determining oocyte quality. In conclusion, our data identified a few miRNAs in the follicular fluid and in the ooplasm that modulate the oocyte developmental competence. This provides new insights that could help with the management of cattle reproductive efficiency.

State-of-the-art in reproductive bench science: Hurdles and new technological solutions.

Infertility is a growing issue in modern society, being the fifth highest serious global disability according to the World Health Organization. To study infertility and other reproductive system complications, bench science still relies on 2D and animal studies, which regularly have been criticized due to their inability to mimic the human body. Particular challenges in 2D studies include the inability to mimic fluid dynamics, gametes modulation and their crosstalk, hormonal patterns as well as the low quality and viability of gametes and embryos. Animal models also present other drawbacks, namely the absence of menstruation, making it difficult to establish a reliable predictive model for the human system. Additionally, reproductive studies should not be limited to the fallopian tube as the sole responsible for most infertility cases, but instead the research spectrum should be widened to the whole reproductive system given the tight interconnectivity between each and every organ. In the last few decades, new in vitro technologies have been developed and applied to the study of reproductive system complications. These systems allow to create complex three-dimensional structures, which are therefore able to more closely resemble specific microenvironments and provide more realistic physical and biochemical cues. 3D (bio)printing, organoids and organs-on-chips are some of the dynamic technologies which are replacing conventionally employed static 2D culture. Herein, we provide an overview of the challenges found in conventional 2D and animal models of the reproductive system and present potential technological solutions for those same challenges.

Evolution of pig intestinal stem cells from birth to weaning.

Pig intestinal epithelium undergoes a complete renewal every 2 to 3 days that is driven by intestinal stem cells (ISCs) located at the crypt base in their niche. Intestinal stem cells generate a pool of highly proliferative transit-amplifying cells, which either migrate up the villus and differentiate into enterocytes and secretory cells or migrate towards the base of the crypt where they differentiate into Paneth cells that secrete antimicrobial peptides. The balance between ISCs' self-renewal and differentiation controls intestinal epithelial homeostasis; therefore, ISCs are essential for ensuring intestinal epithelial integrity. Detailed knowledge of these mechanisms in pig and other domestic species is very limited. Therefore, the aim of this work was to characterize ISC from birth to weaning. We analysed the duodenum, jejunum and colon of six piglets at birth, 6-day-old nursing piglets and 28-day-old weanlings, one week after weaning. We immunolocalized homeobox only protein+ (HOPX) and sex-determining region Y-box 9+ (SOX9) cells that identify quiescent and active ISC, respectively. The volume of ISCs was quantified with stereological methods and was compared to that of mitotic cells expressing proliferating cell nuclear antigen and apoptotic cells identified by the presence of cleaved caspase-3. Furthermore, we compared all these values with crypts and villi measurements and their ratio. Our results indicated that both quiescent and active ISCs are present in pig intestine from birth to weaning and are localized in the crypts of the small and large intestine. However, both markers were also observed along the villi and on the colon luminal epithelium, suggesting that at these stages, pig mucosa is still immature. Weaning induced a dramatic reduction of both HOPX+ and SOX9+ cells, but SOX9+ cells underwent a significantly greater reduction in the small intestine than in the colon. This suggests that the two ISC types are differentially regulated along the intestinal tracts. Overall, the pig ISC complex has many similarities with its murine counterpart, but also has some differences. These include active ISC not showing the typical columnar base morphology as well as the absence of bona fide Paneth cells. This is the first description of ISC dynamics during pig's early life and provides useful reference data for future studies, aimed at targeting ISC for the development of efficient alternatives to in-feed antibiotics for preserving intestinal integrity.

The quest for an effective and safe personalized cell therapy using epigenetic tools.

In the presence of different environmental cues that are able to trigger specific responses, a given genotype has the ability to originate a variety of different phenotypes. This property is defined as plasticity and allows cell fate definition and tissue specialization. Fundamental epigenetic mechanisms drive these modifications in gene expression and include DNA methylation, histone modifications, chromatin remodeling, and microRNAs. Understanding these mechanisms can provide powerful tools to switch cell phenotype and implement cell therapy. Environmentally influenced epigenetic changes have also been associated to many diseases such as cancer and neurodegenerative disorders, with patients that do not respond, or only poorly respond, to conventional therapy. It is clear that disorders based on an individual's personal genomic/epigenomic profile can rarely be successfully treated with standard therapies due to genetic heterogeneity and epigenetic alterations and a personalized medicine approach is far more appropriate to manage these patients. We here discuss the recent advances in small molecule approaches for personalized medicine, drug targeting, and generation of new cells for medical application. We also provide prospective views of the possibility to directly convert one cell type into another, in a safe and robust way, for cell-based clinical trials and regenerative medicine.

Epigenetic conversion of adult dog skin fibroblasts into insulin-secreting cells.

Diabetes is among the most frequently diagnosed endocrine disorder in dogs and its prevalence continues to increase. Medical management of this pathology is lifelong and challenging because of the numerous serious complications. A therapy based on the use of autologous viable insulin-producing cells to replace the lost ß cell mass would be very advantageous. A protocol to enable the epigenetic conversion of canine dermal fibroblasts, obtained from a skin biopsy, into insulin-producing cells (EpiCC) is described in the present manuscript. Cells were briefly exposed to the DNA methyltransferase inhibitor 5-azacytidine (5-aza-CR) in order to increase their plasticity. This was followed by a three-step differentiation protocol that directed the cells towards the pancreatic lineage. After 36 days, 38 ± 6.1% of the treated fibroblasts were converted into EpiCC that expressed insulin mRNA and protein. Furthermore, EpiCC were able to release insulin into the medium in response to an increased glucose concentration. This is the first evidence that generating a renewable autologous, functional source of insulin-secreting cells is possible in the dog. This procedure represents a novel and promising potential therapy for diabetes in dogs.

Phenotype switching through epigenetic conversion.

Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a 'highly permissive' state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed 'epigenetic conversion'. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.

Expression and intracytoplasmic distribution of staufen and calreticulin in maturing human oocytes.

PURPOSE: In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated. METHODS: We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy. RESULTS: STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle. CONCLUSIONS: The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.

Research with parthenogenetic stem cells will help decide whether a safer clinical use is possible.

The derivation and use of parthenogenetic stem cells (pESCs) are envisaged as a reliable alternative to conventional embryonic stem cells. Similar to embryonic stem cells in their proliferation, expression of pluripotency markers and capacity to multilineage differentiation, pESCs are at a lower risk of immune rejection within stem cell-based therapeutics. Moreover, pESCs represent an important model system to study the effect of paternally imprinted genes on cell differentiation. However, currently available information about the genetic and epigenetic behaviour of pESCs is limited. Thus, a detailed look at the biology of parthenogenetic (PG) embryos and PG-derived cell lines would allow gaining insight into the full potential of pESC in biotechnology. In this commentary article we review some features related to the biology of PG embryos and pESCs. In addition, novel traits on bovine pESCs (bpESCs) are discussed.

Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine.

Large animal models provide useful data for pre-clinical research including regenerative medicine. However whereas the derivation of tissue specific stem cells has been successful. pluripotent stem cells so far have been difficult to obtain in these species. A possible alternative could be direct reprogramming but this has only been described in mouse and human. We have recently described an alternative method for reprogramming human somatic cells based on a brief demethylation step immediately followed by an induction protocol. Aim of the present paper was to determine whether this method is applicable to pig in the attempt to achieve cell reprogramming in a large animal model for the first time. Pig dermal fibroblasts were exposed to DNA methyltransferase inhibitor 5-aza-cytidine (5-aza-CR) for 18 h. After a brief recovery period, fibroblast were subjected to a three-step protocol for the induction of endocrine pancreatic differentiation that was completed after 42 days. During the process pig fibroblast rapidly lost their typical elongated form and gradually became organized in a reticular pattern that evolved into distinct cell aggregates. After a brief expression of some pluripotency genes, cells expression pattern mimicked the transition from primitive endoderm to endocrine pancreas. Not only converted cells expressed insulin but were able to release it in response to a physiological glucose challenge in vitro. Finally they were able to protect recipient mice against streptozotocin-induced diabetes. This work shows, that the conversion of a somatic cell into another, even if belonging to a different germ layer, is possible also in pig.

Beneficial effect of directional freezing on in vitro viability of cryopreserved sheep whole ovaries and ovarian cortical slices.

STUDY QUESTION: Does directional freezing improve the structural and functional integrity of ovarian fragments compared with conventional slow freezing and to whole ovary cryopreservation? SUMMARY ANSWER: Compared with slow freezing, the use of directional freezing significantly improves all structural and functional parameters of ovarian fragments assessed in vitro and, overall, whole ovaries were better preserved than ovarian fragments. WHAT IS KNOWN ALREADY: Directional freezing has been developed to provide an alternative way to cryopreserve large biological samples and it is known to improve the structural and functional integrity of whole ovaries. Conventional slow freezing of ovarian fragments is the procedure more widely used in clinical settings but it causes substantial structural damage that limits the functional period after transfer back into the patient. STUDY DESIGN, SIZE, DURATION: We performed a 2 × 2 factorial design experiment on a total of 40 sheep ovaries, divided into four groups (n = 10 ovaries per group): (i) directional freezing of whole ovary (DFwo); (ii) directional freezing of ovarian fragments (DFof); (iii) conventional freezing of whole ovary (CFwo); (iv) conventional freezing of ovarian fragments (CFof). An additional eight ovaries were used as fresh controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ewe ovaries were randomly assigned to one of the experimental groups and frozen accordingly. Upon thawing, ovarian tissue was examined morphologically and cultured in vitro for 7 days. Samples were analyzed for cell proliferation and apoptosis, for DNA damage and repair activity, and for the presence of a panel of heat shock proteins (HSPs) by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: Most studied parameters were significantly improved (P < 0.05) in all samples cryopreserved with directional compared with slow freezing. The proportion of primordial follicles, which developed to the primary stage in whole ovaries (53 ± 1.7%) and in ovarian fragments (44 ± 1.8%) cryopreserved with directional freezing, was greater than with slow frozen whole ovaries (6 ± 0.5%, P = 0.001) or fragments (32 ± 1.5%, P = 0.004). After 7 days of culture, cell proliferation in DFwo (28 ± 0.73%) was the highest of all groups (P < 0.05) followed by DFof (23 ± 0.81%), CFof (20 ± 0.79%) and CFwo (9 ± 0.85%). Directional freezing also resulted in a better preservation of the cell capacity to repair DNA damage compared with slow freezing both in whole ovaries and ovarian fragments. Apoptosis and HSP protein levels were significantly increased only in the CFwo group. Direct comparison demonstrated that, overall, DFwo had better parameters than DFof and was no different from the fresh controls. LIMITATIONS, REASONS FOR CAUTION: The study is limited to an in vitro evaluation and uses sheep ovaries, which are smaller than human ovaries and therefore may withstand the procedures better. WIDER IMPLICATIONS OF THE FINDINGS: Improved integrity of ovarian morphology may translate to improved outcomes after transplantation. Alternatively, the particularly good preservation of whole ovaries suggests they could provide a source of ovarian follicles for in vitro culture in those cases when the presence of malignant cells poses a substantial risk for the patient. STUDY FUNDING/COMPETING INTEREST(S): Supported by: Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 10376, Carraresi Foundation and by Legge 7 Regione Autonoma Sardegna (R.A.S). There are no conflicts of interest.

Chronic mastitis is associated with altered ovarian follicle development in dairy cattle.

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3 mm and 4 to 7 mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8 mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity.

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.

Large animal models for cardiac stem cell therapies.

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.

No shortcuts to pig embryonic stem cells.

The establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines. These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC. Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells.

Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins.

In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at approximately 60-90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 +/- 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins.

Parthenogenetic activation: biology and applications in the ART laboratory.

Parthenogenesis is a reproductive strategy typical of lower species where a female gives birth to offsprings without a paternal contribution. On the contrary, parthenogenesis is not a form of natural reproduction in mammals even if mammalian oocytes, under appropriate stimuli, can undergo to parthenogenetic activation. This review describes the biological mechanisms regulating parthenogenetic activation in mammals and illustrates the fundamental differences between embryos and parthenotes. Ethical, legal and political concerns on the value of human embryos regulate and limit human embryological studies founded on the widespread belief that human embryos should not be created and studied for research purposes only. Based on the differences between parthenotes and embryos the use of parthenogenesis is proposed as an experimental tool to investigate embryo development which may solve many of the ethical concerns associated with the use of human embryos for experimental purposes. Examples of the possible uses of parthenotes in many field of research such as in vitro assays aimed to study some aspects of assisted reproductive technologies (ART), toxicology or stem cell are described and their validity is discussed.

Recent progress in embryonic stem cell research and its application in domestic species.

Many reports described cell lines derived in domestic species, which presented several important features typical of embryonic stem cells (ESCs). Such features unfortunately did not include the capacity to generate germ-line chimeras, therefore limiting the possibility to use these cells as tools for the genetic manipulation. However, farm animal ESCs may still be useful for the generation of transgenic animals as usually have a self-renewal capacity more prolonged than normal primary cultures thus increasing the possibility to transform and select cells to be used as nucleus donors in cloning procedures. Farm animal ESCs may also be an excellent experimental model in pre-clinical trials, assessing the feasibility of cell therapy because of the close morphological and physiological resemblance to humans of species like the pig. However, the persistent lack of standard methods for the derivation, maintenance and characterization of ESCs in domestic species stimulated the search for alternatives. Embryonic germ cells may represent such an alternative. Indeed, these cells showed a higher plasticity than ESCs as contributed to embryonic development forming chimeric newborns but, as for ESCs, standardization is still far away and efficiency is very low. Recent results indicated spermatogonial stem cells as possible tools for germ-line genetic modifications with some proof of principle results already achieved. But, a real break through could arrive from the multipotent germ-line stem cells, virtually equivalent to ESC, derived from newborn and adult mouse testis.