Artificial activation of ovine oocytes is required after ICSI with freeze-dried spermatozoa.

Objectives. Freeze-drying allows to store the biological samples in a dry state and represents an interesting alternative low-cost strategy of semen biobanking to save the endangered species. Here, we have established a dry sperm biobank from an endangered Italian sheep breed (Pagliarola) and tested its efficiency through ICSI. Materials and methods. The motile spermatozoa from ram ejaculates collected with artificial vagina was selected by swim-up in TRIS-based medium (2.42g TRIS, 1.36g citric acid, 1.00g fructose, 100.000 U.I. penicillin G, 100mg streptomycin, in 67.20ml bidistilled water (ddH2O); pH was adjusted to 6.7) for 20 minutes at 38.5°C. The motile spermatozoa were frozen in freeze-drying medium (10mM EGTA and 50mM NaCl in 10mM Tris–HCl buffer; pH was adjusted to 8.4) in a -80°C freezer for 75 minutes and subsequently lyophilized by the freeze-drying apparatus SP Scientific-VirTis, Freeze-dryer 2.0 BenchTop, 20 hours with a condenser temperature of -58°C and vacuum of 20 mTorr). The vials were sealed in glass vials under vacuum and stored in the dark at 4°C for 1-2 months. Just before the ICSI, the freeze-dried spermatozoa were rehydrated by adding 100µl ddH2O. To evaluate the fertilizing capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI and allocated to two groups: 56 oocytes were activated by incubation with 5μM ionomycin (ICSI-FDSa); 52 were left un-activated (ICSI-FDSna). Forty-four oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control. Pronuclear formation (2PN) and blastocyst development were investigated at 14-16 hours and 7-8 days after ICSI, respectively. Differences were considered statistically significant for p<0.05 (Chi-square test). Data were analyzed using PRISM, software version 5.0; GraphPad. Results. The freeze-dried spermatozoa were completely immotile after rehydration, however they maintained the capacity to fecund oocytes after ICSI. Two PN were found in 83.3% of ICSI-FDSa, 81.4% of ICSI-FS while only in 14.3% of ICSI-FDSna (p<0.05 ICSI-FDSna vs ICSI-FDSa; p<0.01 ICSI-FDSna vs ICSI-FS). Likewise, the ICSI freeze-dried spermatozoa yielded blastocysts only following artificial activation (ICSI-FDSa: 10.2%; ICSI-FS: 31%; ICSI-FDSna: 0%; p<0.05 ICSI-FDSa vs ICSI-FDSna andICSI-FS; p<0.0001 ICSI-FDSna vs ICSI-FS). Conclusions. Our finding show that freeze-dried spermatozoa have lost the capacity to trigger oocyte activation but maintained their nuclear viability, whose developmental potential was fully released following artificial activation. Our results support the evidence that freeze-drying effective approach of spermatozoa storage to save endangered species

Freeze-dried sperm: an alternative biobanking solution for endangered farm species

Introduction An ever-increasing number of domestic species are threatened with extinction. Biobanking of spermatozoa could represent a feasible and efficient way for preserving genetic heritage and to maintain biodiversity. Given the published evidence that lyophilized spermatozoa retain their fertilizing capacity, we have collected semen from an Italian endangered sheep breed (Pagliarola) and created a biobank of cryopreserved and freeze-dried spermatozoa. Material and Methods The fertilizing capacity of the all stored semen, cryopreserved and freeze-dried, was evaluated by IVF and ICSI, respectively. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were un-activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16 hours after ICSI in fixed presumptive zygotes. Results and Discussion As expected, the fertilizing capacity of cryopreserved Pagliarola’s spermatozoa was comparable to commercial semen stocks (31.8% vs 29%, respectively). Two PN were observed in 83.3% of ICSI-FDSa while only in 14.3% of ICSI-FDSna. Likewise, only artificially activated oocytes were able to develop to blastocyst after ICSI (10.2% compare to 0% with ICSI-FDSna). Oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control (81.4% of 2PN; 31% of blastocyst). In this work, we have demonstrated for the first time that freeze-dried ram spermatozoa could drive blastocyst development following ICSI. Although the developmental potential of embryos derived from lyophilized spermatozoa was significantly lower than cryopreserved ones, sperm lyophilization may be an alternative, low cost storage option, susceptible of improvement of course, to save biodiversity in domestic species

Freeze-dried spermatozoa: An alternative biobanking option for endangered species

In addition to the iconic wild species, such as the pandas and Siberian tigers, an ever-increasing number of domestic species are also threatened with extinction. Biobanking of spermatozoa could preserve genetic heritages of extinct species, and maintain biodiversity of existing species. Because lyophilized spermatozoa retain fertilizing capacity, the aim was to assess whether freezedried spermatozoa are an alternative option to save endangered sheep breeds. To achieve this objective, semen was collected from an Italian endangered sheep breed (Pagliarola), and a biobank of cryopreserved and freeze-dried spermatozoa was established, and evaluated using IVF (for frozen spermatozoa) and ICSI procedures (for frozen and freeze-dried spermatozoa). As expected, the fertilizing capacity of cryopreserved Pagliarola’s spermatozoa was comparable to commercial semen stocks. To evaluate the activating capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI, and allocated to two groups: 56 oocytes were activated by incubation with ionomycin (ICSI-FDSa) and 52 were not activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14–16 h after ICSI in fixed presumptive zygotes. Only artificially activated oocytes developed into blastocysts after ICSI. In the present study, freeze-dried ram spermatozoa induced blastocyst development following ICSI at a relatively high proportion, providing evidence that sperm lyophilization is an alternative, low cost storage option for biodiversity preservation of domestic species

Alternative strategies for nuclear reprogramming in somatic cell nuclear transfer (SCNT)

Twenty years passed by since the production of Dolly the sheep, but despite significant technical progress has been achieved in the manipulation procedures, the proportion of offspring following transfer of SCNT embryos has remained almost unchanged in farm animals. Remarkable progress has been obtained instead in laboratory animals, particularly by Japanese Groups, in the mouse. However, the nuclear reprogramming strategies tested in mouse do not always work in farm animals, and others are difficult to be implemented, for require complicated molecular biology tools unavailable yet in large animals. In this review we put in contest the previous work done in farm and laboratory animals with recent achievements obtained in our laboratory, and we also indicate a road map to increase the reliability of SCNT procedures

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

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

Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer

Nuclear quiescence and histone hyper-acethylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts.

Recently we demonstrated the possibility of the direct histone-protamine exchange in somatic cells by the exogenous induction of protamine 1 (Prm1) gene expression. Here we have further advanced our protocol, by mimicking the nuclear remodelling taking place in spermatogenesis. The spermiogenesis starts by cell quiescence and the open of nuclear chromatin (by histone-hyperacetylation) of post-meiotic round spermatids. Our aim was to test if protaminization of somatic nucleus increases through the correct union of the induction of G0 stage and exposure to Trichostatin A (TSA). To stop the cell proliferation, the cells were cultured in 0.5% FBS in MEM (G0 group) whereas in the control group (CTR) the cells were culture in 10% FBS in MEM. To check the proper TSA concentration, we treated the cells with 25 nM (25 TSA), 50 nM (50 TSA) and 100 nM (100 TSA) in MEM + 10% FBS. Our results showed that combination of the cell culture condition in G0 and 50 TSA stopped the cell proliferation vs CTR (respectively 17.8%, 8% and 90.2%, p<0.0001). Moreover, quiescent markers gene expression analysis (Dicer1, Smarca 2, Ezh1, Ddx39, H2afz and Pink1) demonstrated that our cell culture condition drive the cell in a quiescent state. Finally, the union of G0 and 50 TSA produced a higher number of spermatid-like cell (39.4%) than the 25 TSA (20.4%, p<0.05) and 100 TSA (13.7%, p<0.05). To conclude, we have demonstrated that the open chromatin structure conferred by G0 stage and TSA, resulted in a more efficient Prm1-mediated conversion of somatic nuclei into spermatid-like structures

A New, Dynamic Era for Somatic Cell Nuclear Transfer?

Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to ‘rescue’ endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs)