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

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 < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential

THE SPERM ASTER NUCLEATION AND MICROTUBULE ELONGATION IN IN VITRO FERTILIZED SHEEP ZYGOTES

Successful fertilization process and embryo development relies on functional centrioles/centrosomes which have been confirmed to be paternally inherited in most farm animals, including sheep.2,3,4 Shortly after fertilization, the sperm proximal centriole typically nucleates microtubular aster that function as microtubule organizing center and ensure paternal and maternal genomes merging.5,6,7 At the moment, there are no data on the timing and dynamics of sperm aster organization in sheep. In this study, we have traced the fate of sperm centriole after fertilization to evaluate the timing of the sperm microtubular aster nucleation in early sheep zygotes. To this extend, we have imaged sperm aster nucleation at different post-fertilization moments throughout α-tubulin immunofluorescence in early in vitro fertilized sheep oocytes. To visualize the process of sperm aster nucleation as well as microtubules elongation, sheep oocytes were subjected to in vitro maturation (IVM) for 24 h followed by in vitro fertilization (IVF). IVF was performed in 50 μl drops of synthetic oviductal fluid (SOF) with estrus sheep serum and 16 μM isoproterenol, covered by mineral oil. In a preliminary experiment, we have established that spermatozoon takes almost 3 hours to complete the fertilization and to enter the oocyte. Fertilization has been arrested at different timing after sperm-egg co-culture (from 4 up to 7 hours) and then presumptive zygotes have been removed from zona pellucida, fixed and examined with anti-α-tubulin immunofluorescence under confocal microscopy. We have observed that the sperm centriole initiates sperm aster nucleation within 1 hour post-fertilization (4h from sperm-egg co-culture) and that microtubules elongation takes place approximately 3 hours post-insemination. Future investigations will aim to identify which sperm centriole contributes to embryonic inheritance

Reducing the time of spermatozoa/oocyte interaction improves embryonic development in sheep.

In vitro fertilization (IVF) is a well-established Assisted Reproductive Technique (ART) routinely applied in human infertility treatment and in large animals, both to boost reproductive performances of selected genotypes, and also for basic research. Despite its value, IVF has seen very little progress in the last two decades, and relies to established paradigms, like overnight sperm-egg co-incubation. However, the long exposure to the relatively high spermatozoa concentration in a dish increases the risk of polyspermy and could be detrimental for early embryonic development. In our work we have identified the time window within which the fertilization occurs in order to refine the procedure and reduce sperm-egg co-incubation, comparing embryo development after short (sIVF) and overnight spermatozoa/oocyte co-incubation (o/nIVF). A total of 144 in vitro matured sheep oocytes were co-incubated with spermatozoa in IVF medium [synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16µM isoproterenol]. Then, small batches of oocytes were collected every 30 minutes to check for the presence of a fertilizing spermatozoon. To assess that, 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. Pronuclear formation (PN) and embryo development were evaluated after 16 hours (PN), 24 hours (2 cells) and 7 days of culture (blastocyst). We found that spermatozoa engaged with the ZP not earlier than 90 minutes and penetrated the oocytes within 3-4 hours after IVF. A trend for a lower polyspermic fertilization (>2PN) was detected in sIVF (7.7%) vs o/nIVF (18.4%,). Likewise, cleavage and blastocyst rate were higher after sIVF compared to o/n-IVF (2-cells: 38.6% vs 23.8%; blastocyst: 18.1% vs 9.5%; sIVF vs o/nIVF). This work demonstrates that 4 hours spermatozoa/oocyte interaction are sufficient to achieve fertilization, reduce polyspermy and improve early embryonic development in sheep