Comparison of slow and rapid freezing for long term storage of freeze-dry ram spermatozoa

Semen lyophilization is an interesting technique that might be a cheap alternative to long-term storage under liquid nitrogen. The first significant result of this method was achieved by Wakayama and Yanagimachi in the 1998 [1] demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. From this work on, the most used approach for lyophilisation is that of deep-freezing, that is directly immersing the semen sample into liquid nitrogen before vacuum drying. Recently we have shown that it is possible to establish a "dry" bank of ejaculated and epidydimal freeze-dried ram spermatozoa [2, 3]. In order to improve and make the technique more reliable, here we focused on the freezing phase, comparing two different protocols: i) Fast-freezing, where the semen is plunged directly into liquid nitrogen (LN group); ii) Slow-freezing, where the sample is progressively cooled to a final temperature of -50°C (SL group). Briefly, for the preparation of the LN group sample the protocol reported in [2] was followed, while for the SL group the semen was frozen with a freezing rate of 1°C/min until -50°C degrees, when the sample was placed inside the lyophilizer. Dry spermatozoa from both groups was used for Intracytoplasmic Sperm Injection (ICSI) and the embryo development was evaluated at 24h (2-Cells stage) and 7 days (expanded blastocyst) after fertilization. At 24h post fertilization the SL-group showed a higher number of cleaved embryos than LN-group (42/100 (42%) versus 19/75 (25.3%), P=0.0253, SL and LN respectively). At 7 days after fertilization the blastocyst rate in SL-group was higher (7/100 (7%)) than in LN-group (2/75 (2.7%)), although not statistically different. Our data shows that lyophilisation can be conveniently achieved in ram spermatozoa without previous freezing in liquid nitrogen, thus simplifying the procedure. This data supports the idea that lyophilisation might be a valuable and cheaper alternative to liquid nitrogen for long-term storage of ram semen

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

Evidence of placental autophagy during early pregnancy after transfer of in vitro produced (IVP) sheep embryos

Pregnancies obtained by Assisted Reproductive Technologies (ART) are associated with limited maternal nutrient uptake. Our previous studies shown that in vitro culture of sheep embryos is associated with vascularization defects in their placentae and consequent reduction of embryo growth. Autophagy is a pro-survival cellular mechanism triggered by nutrient insufficiency. Therefore, the goal of our present study was to determine if autophagy is involved in early placental development after transfer of in vitro produced (IVP) embryos. To do this, placentae obtained following transfer of IVP sheep embryos were compared with placentae obtained after natural mating (control-CTR). The placentae were collected on day 20 post-fertilization and post-mating, respectively, and were analyzed using molecular (qPCR), ultrastructural and histological/immunological approaches. Our results show drastically increased autophagy in IVP placentae: high levels of expression (p<0.05) of canonical markers of cellular autophagy and a high proportion of autophagic cells (35.08%; p<0.001) were observed. We conclude that high autophagic activity in IVP placentae can be a successful temporary counterbalance to the retarded vasculogenesis and the reduction of foetal growth observed in pregnancies after transfer of IVP embryos

Population Saturation in Trivalent Erbium Sensitized by Organic Molecular Antennae

We investigate sensitization efficiency of near-infrared emission and population saturation of trivalent erbium in erbium quincilinolato complexes photoexcited into the absorption band of the organic sensitizer. At low excitation levels, we find high (similar to 80%) sensitization efficiencies. We observe excited state population saturation at inversion threshold under subnanosecond pumping at the level of one injected photoexcitation per complex

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

Synergies between assisted reproduction technologies and functional genomics

This review, is a synopsis of advanced reproductive technologies in farm animals, including the discussion of their limiting factors as revealed by the study of offspring derived from embryos produced in vitro and through cloning. These studies show that the problems of epigenetic mis-programming, which were reported in the initial stages of assisted reproduction, still persist. The importance of whole-genome analyses, including the methylome and transcriptome, in improving embryo biotechnologies in farm animals, are discussed. Genome editing approaches for the improvement of economically-relevant traits in farm animals are also described. Efficient farm animal embryo biotechnologies, including cloning and the most recent technologies such as genome editing, will effectively complement the latest strategies to accelerate genetic improvement of farm animals

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