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

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

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

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

Dry biobanking as a conservation tool in the Anthropocene

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity – humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry – innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach

Programming of embryonic development

Assisted reproductive techniques (ART) and parental nutritional status have profound effects on embryonic/fetal and placental development, which are probably mediated via “programming” of gene expression, as reflected by changes in their epigenetic landscape. Such epigenetic changes may underlie programming of growth, development, and function of fetal organs later in pregnancy and the offspring postnatally, and potentially lead to long-term changes in organ structure and function in the offspring as adults. This latter concept has been termed developmental origins of health and disease (DOHaD), or simply developmental programming, which has emerged as a major health issue in animals and humans because it is associated with an increased risk of non-communicable diseases in the offspring, including metabolic, behavioral, and reproductive dysfunction. In this review, we will briefly introduce the concept of developmental programming and its relationship to epigenetics. We will then discuss evidence that ART and periconceptual maternal and paternal nutrition may lead to epigenetic alterations very early in pregnancy, and how each pregnancy experiences developmental programming based on signals received by and from the dam. Lastly, we will discuss current research on strategies designed to overcome or minimize the negative consequences or, conversely, to maximize the positive aspects of developmental programming

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

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