The impaired development of sheep ICSI derived embryos is not related to centriole dysfunction

While intracytoplasmic sperm injection (ICSI) is an asset in human Assisted Reproduction Technologies (ART), its outcomes, in terms of blastocyst, is still unacceptably low in ruminants. The picture typically found in ICSI derived bovine and ovine embryos is an asymmetry between a high activation rate, marked by a pronuclear development, and a low first cleavage rate. Abnormal centriole function has been indicated as a possible factor which undermines embryonic development following ICSI, especially when Freeze Dried spermatozoa (FD) are used. In order to verify the hypothesis that centriole dysfunction might be responsible for low ICSI outcomes in sheep, we have investigated micro-tubular dynamics, markedly aster nucleation, in fertilized sheep zygotes by ICSI with frozen/thawed (FT) and FD spermatozoa; In Vitro Fertilized (IVF) sheep oocytes were used as control. The spermatozoa aster nucleation was assessed at different time points following ICSI and IVF by immune-detection of α-tubulin. Pronuclear stage, syngamy and embryo development were assessed. No difference was noticed in the timing of aster nucleation and microtubule elongation in ICSI-FT derived embryos with control IVF ones, while a delay was recorded in ICSI-FD ones. The proportion of 2-pronuclear stage zygotes was similar in ICSI-FT and ICSI-FD (47% and 53%, respectively), both much lower comparing the IVF ones (73%). Likewise, syngamy was observed in a minority of both ICSI groups (28.5% vs 12.5% in ICSI-FT/FD respectively) comparing to IVF controls (50%), with a high number of zygotes blocked at the 2-pronuclear stage (71.5% vs 87.5% respectively). While no significant differences were noticed in the cleavage rate between ICSI-FD, ICSI-FT and IVF groups (31%, 34% and 44%) respectively, development to blastocyst stage was markedly compromised in both ICSI groups, especially with FD spermatozoa (10% in ICIS-FD and 19% in ICSI-FT vs 33% in IVF (P < 0.005, ICSI-FD vs IVF and P < 0.05, IVF vs ICSI-FT, respectively). Hence, here we have demonstrated that the reduced cleavage, and the ensuing impaired development to blastocysts stage of ICSI derived sheep embryos is not related to centriole dysfunction, as suggested by other authors. The major recorded problem is the lack of syngamy in ICSI derived zygotes, an issue that should be addressed in further studies to improve ICSI procedure in sheep embryos

Somatic cell nuclear transfer: failures, successes and the challenges ahead

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before

PPARγ2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPARγ2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPARγ2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPARγ2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPARγ2 is a powerful modulator of stem cell-related gene expression. In general, PPARγ2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPARγ agonist TZD rosiglitazone specifically affects the expression of “stemness” genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPARγ2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPARγ2 role in stem cell biology

Ultrastructural analysis reveals abnormal mitochondria in cloned blastocysts

Somatic cell nuclear transfer (SCNT) is a powerful technique, but still very inefficient despite 20 years passed by since the cloned mammal was born. We have recently shown that the major cause of abnormalities observed in cloned fetuses are mitochondrial dysfunctions in placenta collected from cloned sheep. Investigations on mitochondria in SCNT are limited to the mtDNA hetero/homoplasmy in cloned offspring, whereas no data is available for an eventual role of mitochondria dysfunction on the developmental failure of cloned animals. Here we wanted to know whether mitochondrial abnormalities are observed already in cloned blastocysts since mitochondrial replication does not occur after the hatched blastocysts stage. SCNT and in vitro processed (IVP) blastocysts were produced and analysed for mitochondrial structure and functionality. First, embryos were analysed using transmission electron microscope (TEM). Drastic differences in mitochondrial structure between SCNT and IVP blastocysts were observed. Decrease density of mature mitochondria, very high degree of cytoplasmic vacuolisation, numerous cytoplasmic vesicle and autophagosomes were observed in SCNT blastocysts. Moreover, statistically lower expression of major mitochondrial, autophagic and apoptotic proteins were observed in SCNT embryos. Obtained results clearly shown that mitochondrial abnormalities are already observed in blastocysts stage embryos. It is important to point out that activity of mitochondria are strictly control by nuclear signals, thus, obtained results may suggest that incomplete nuclear reprogramming in cloned nucleus might be responsible also for the impaired mitochondrial function in cloned embryos/fetuses

Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions

This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar ‘intrinsically disordered’ proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa

Controlled spermatozoa–oocyte interaction improves embryo quality in sheep

The current protocols of in vitro fertilization and culture in sheep rely on paradigms established more than 25 years ago, where Metaphase II oocytes are co-incubated with capacitated spermatozoa overnight. While this approach maximizes the number of fertilized oocytes, on the other side it exposes them to high concentration of reactive oxygen species (ROS) generated by active and degenerating spermatozoa, and positively correlates with polyspermy. Here we set up to precisely define the time frame during which spermatozoa effectively penetrates and fertilizes the oocyte, in order to drastically reduce spermatozoa-oocyte interaction. To do that, in vitro matured sheep oocytes co-incubated with spermatozoa in IVF medium were sampled every 30 min (start of incubation time 0) to verify the presence of a fertilizing spermatozoon. Having defined the fertilization time frame (4 h, data from 105 oocytes), we next compared the standard IVF procedures overnight (about 16 h spermatozoa/oocyte exposure, group o/nIVF) with a short one (4 h, group shIVF). A lower polyspermic fertilization (> 2PN) was detected in shIVF (6.5%) compared to o/nIVF (17.8%), P < 0.05. The o/nIVF group resulted in a significantly lower 2-cell stage embryos, than shIVF [34.6% (81/234) vs 50.6% (122/241) respectively, P < 0.001]. Likewise, the development to blastocyst stage confirmed a better quality [29% (70/241) vs 23.5% (55/234), shIVF vs o/nIVF respectively] and an increased Total Cell Number (TCN) in shIVF embryos, compared with o/n ones. The data on ROS have confirmed that its generation is IVF time-dependent, with high levels in the o/nIVF group. Overall, the data suggest that a shorter oocyte-spermatozoa incubation results in an improved embryo production and a better embryo quality, very likely as a consequence of a shorter exposure to the free oxygen radicals and the ensuing oxidative stress imposed by overnight culture

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120 days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60 days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies

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