MICROTUBULAR NUCLEATING CENTER DYNAMICS IN ASSISTED REPRODUCTION TECHNOLOGY (ICSI, SHEEP MODEL)

The scope of this thesis is to investigate abnormal sperm centriole function during the early stages of Intra Cytoplasmic Sperm Injection (ICSI) embryo development using sheep as an animal model. The main focus was on evaluating the timing and dynamics of the sperm mi-crotubular aster nucleation and organization as a possible factor that undermines early em-bryonic development following ICSI. The main finding was that ICSI derived embryos using freeze-dried spermatozoa displayed a delay in their aster nucleation and a noticeable ham-pered embryo development. We also noticed that ICSI-derived embryos failed to undergo subsequent development and were blocked at the pronuclear stage. In this work, we demon-strated that embryo development failure following ICSI in sheep is not actually related to a centriole dysfunction; rather, the major problem recorded is the lack of syngamy. Thus, besides our objective data ruling out centriole dysfunction as a cause of developmental fail-ure in sheep/ruminant embryos, we have opened a worth theme to investigation, that is the perfecting of artificial protocols in sheep oocytes fertilized by ICSI

Monitoring the microtubule nucleation dynamics of sperm centriole after IFV and ICSI in sheep zygotes.

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive (ART) technique that is less efficient in ruminant, comparing to other species. In mammals, the spermatozoa proximal centriole nucleates the microtubule and generates the functional cell centriole of the resulting organism. Our study aimed to investigate eventual difference in the centriole microtubular nucleation in ICSI fertilized oocytes, comparing to control In Vitro Fertilized ones (IVF). In fact, we made the hypothesis that the tail severing step achieved in our ICSI protocol through applying a few piezo pulses, might mechanically damage the proximal centriole. On this basis, Sheep oocytes were in vitro maturated (IVM) for 24 h then were injected by piezo-pulsed spermatozoa, chemically activated by 5 min of incubation with 5 mg/ml ionomycin, washed in H199 for 5 min and cultured in 50 µl drops of Synthetic Oviductal Fluid (SOF) with estrus sheep serum and 16 µM isoproterenol, covered by mineral oil. Fertilization has been arrested around 5h after ICSI, and the presumptive zygotes were processed for immunological detection of tubulin. Zona Pellucida (ZP) was removed with a combined treatment of acid Tyrode and trypsin and zygotes were then fixed with 4% paraformaldehyde (pH7.2) and permeabilized by 0.5% Triton X-100, for 20 min each. Microtubular nucleation was assessed with anti-α-tubulin immunofluorescence under confocal microscopy. No difference was noticed in the dynamics and timing of sperm microtubular aster nucleation, that started around 5h post ICSI (5h30). Therefore, we conclude that abnormal microtubular nucleation by the centriole is not responsible for the low development of ICSI fertilized sheep oocytes

Short spermatozoa–oocyte co-incubation improves outcomes of IVF in sheep

The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm–egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm–egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa–oocyte co-incubation. A total of 666 in vitro–matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30 min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16 h (PN), 24 h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24 h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90 min from the beginning of co-incubation and achieve fertilization within 4 h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P = 0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24 h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P = 0.001; blastocyst: 29.4% vs. 20.5%, P = 0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4 h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality

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 a-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

Assessing the effects of bovine embryo-derived extracellular vesicles on the development of individually cultured bovine embryos

In vitro embryo production requires an enriched microenvironment with various vital cell-secreted factors. In vitro cultured single bovine embryos have demonstrated lower blastocyst rate compared to grouped cultured embryos. We assumed that extracellular vesicles (EVs) within an embryo culture system may affect normal in vitro development. This study aimed to assess the supplementation effects of bovine embryo-derived EVs on the development of individually cultured bovine embryos. Bovine oocytes were in vitro maturated (IVM) for 24 h and then in vitro fertilized (IVF). In preliminary experiments, we established that group cultured embryos in EV depleted Bovine Serum Albumin (BSA) media successfully completed their development; while single cultured embryos were only able to reach the morula stage and then degenerated. Hence, we tested EVs supplementation effects in droplets of EV depleted BSA media covered by mineral oil. EVs used for supplementation were produced from single embryos cultured for 8 days in droplets of BSA culture media under mineral oil. Conditioned medium was collected on day 5. EVs were purified, using Izon columns, from embryos which reached the blastocyst stage and embryos which cleaved on day 2 then degenerated. Non-EV supplemented single embryos cultured in BSA media were considered as control. Purified EVs were characterized by nanoparticle tracking analysis and transmission electron microscope (TEM). A total of 8.8 ×106 particles/ml, which we assumed to be the approximate amount of EVs that a single embryo may release during in vitro culture, was supplemented to each droplet on day 4 post-fertilization. Cleavage rates were 70 and 80% for the supplemented groups and 86% for the control. Morula rates were 40%, 47%, and 47% respectively. No blastocyst was observed within the supplemented groups while the control group counted 33% of blastocysts. Our study suggests that BSA EVs support single cultured embryos to complete their development and that a single embryo needs a significant amount of EVs to reach the blastocyst stage. More researches are needed to understand the role of culture media EVs in supporting single embryo development

Individually cultured bovine embryos produce extracellular vesicles that have the potential to be used as non-invasive embryo quality markers

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p Peer reviewe

Characterizing the mitochondrial diversity of Arbi goats from Tunisia

Arbi is one of the main local goat breeds in Tunisia, representing an important economic resource in arid and hot areas where cattle and sheep cannot thrive successfully. In the current work, we have characterized the mitochondrial diversity of 26 Arbi goats by partially sequencing the mitochondrial D-loop region. These sequences plus 10 retrieved from GenBank were analyzed with the DnaSP v.5.10.1, evidencing the existence of 12 different haplotypes. Nucleotide and haplotype diversities were 0.02 and 0.96. Moreover, median-joining network analysis showed that all D-loop sequences from Arbi goats correspond to haplogroup A and that in general they do not cluster with sequences from other goat breeds. The high diversity that has been observed in North African goats is compatible with the maritime diffusion of the Neolithic package 10,000–7000 YBP. Moreover, there are evidences that local Tunisian breeds have been extensively crossed with highly productive transboundary breeds in order to improve meat and milk yields. These uncontrolled crossing practices may lead to the loss of alleles that play key roles in the adaptation of Tunisian local breeds to a harsh environment.This research was funded by the Ministry of Higher Education and Scientific Research of Tunisia. We also acknowledge financial support from the Spanish Ministry of Economy and Competitiveness, through the grant AGL2016-76108-R and the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016–2019 (SEV‐2015‐0533). This research was also supported by the CERCA Programme of the Generalitat de Catalunya.Peer reviewe

Assessing the resistance of sheep fibroblasts to increasing concentration of Trehalose

Trehalose is a non-reducing disaccharide commonly used as a cryo-protectant for deep freezing of cells. In this preliminary work, we have assayed the feasibility of its use for inducing reversible drying in somatic cells (fibroblasts), using sheep as a model. Here we have assayed the tolerance of somatic cells to increasing concentration of trehalose. To this extends, sheep fibroblasts were incubated using a growing concentration gradient of trehalose 50mM, 100mM, 250mM, 400mM and 500mM in Minimum Essential Medium (MEM) for 24hours in an incubator set at 38.5°C. On the basis of preliminary trials, the highest concentrations were excluded because they were found to be toxic to the cells and we have focused on the following: 50mM, 100mM and 200mM. The final osmolarity of the three concentrations were 376 mOsm/kg, 462 mOsm/kg and 711 mOsm/kg respectively. Cells have been incubated in MEM with trehalose for 3 hours in a stove set at 37°C and 12% humidity in lack of gas. Every hour cells were assayed for morphology and viability through Trypan Blue exclusion test. After one hour, live cells were about 71%, 63.15% and 52.85% respectively for 50mM, 100mM and 200mM explaining a negative correlation between the percentage of live cells and the sugar concentration. However, after 2 and 3 hours, death of cells was observed within each concentration as well as morphological changes which was however reversible upon washing cells from trehalose and further culture