μM Ionomycin Research Articles

P-225 Implementing a more physiological method to trigger oocyte activation in cases of extremely poor fertilization with ICSI

Abstract Study question Can we utilize a more physiological compound to induce oocyte activation in couples with history of complete/partial fertilization failure with ICSI? Summary answer Recombinant human phospholipase-C-zeta (rhPLCζ) successfully activated oocytes with same efficiency as ionomycin, consistently normalized fertilization and enhanced clinical outcomes. What is known already While ICSI overcomes most aspects of male gamete dysfunction, partial or complete fertilization failure may seldomly occur mostly due absence of sperm-specific protein, known as PLCζ, which is required to trigger oocyte activation. Assisted oocyte activation (AOA), most often performed with an antibiotic (ionomycin), activates oocytes by triggering calcium release from intracellular storage. This agent induces indiscriminate permeabilization of ooplasmic membrane-bound organelles. This action has been considered nonspecific and may have possible deleterious effects on subsequent embryo development. Therefore, we propose a physiological alternative that employs a recombinant form of PLCζ, the native protein residing in the sperm perinuclear theca. Study design, size, duration In the past 12 months, couples with poor ICSI fertilization were included. PLCζ were assessed to confirm sperm-related oocyte activation deficiency with a normal threshold of ≥ 30%. In a preliminary comparison, sibling oocytes were equally allocated to AOA by ionomycin or to rhPLCζ at time of ICSI. Fertilization, embryo development, and clinical outcomes between the two cohorts. Subsequently, we describe the clinical outcomes of couples treated solely by rhPLCζ. Participants/materials, setting, methods Thirty couples, confirmed with low level of PLCζ in the ejaculates, were offered AOA with their subsequent ICSI cycles (IRB#0712009553). Conventional AOA was performed by exposing post-ICSI oocytes to 50 µM ionomycin. The rhPLCζ-AOA method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5 µg/µL) during ICSI. Chi-square tests were utilized to compare rhPLCζ-AOA versus ionomycin-AOA and rhPLCζ-AOA versus history cycles. Main results and the role of chance In the preliminary study, 9 patients were included (maternal age: 35.7 ± 6, paternal age: 36.7 ± 6). PLCζ positivity was 11.7±3.6% confirming a sperm-related oocyte activation deficiency. A total of 123 oocytes were retrieved in 9 cycles, with maturity of 78.0% (96/123). The 96 MII oocytes were allocated to be treated with ionomycin-AOA (n = 58) or rhPLCζ-AOA (n = 38). While conventional ionomycin-AOA yielded a fertilization rate at 55.2% (32/58), rhPLCζ-AOA resulted in 63.1% (24/38). Both compounds generated embryos at similar rates and one pregnancy was achieve in each cohort. On the bases of such comparable outcomes, 21 couples had 40 previous cycles that generated a fertilization rate of 18.3% (60/327), cleavage rate of 51.7% (31/60), Following the replacement of 23 embryos in 14 transfer cycles, these cycles yielded 2 clinical pregnancies, but all resulted in pregnancy loss. In the subsequent cycles with rhPLCζ-AOA (n = 32), an enhanced fertilization rate was achieved at 41.1% (116/280, P<0.00001). The treatment cycle yielded a higher cleavage rate at 80.2% (93/116, P<0.0001), and after replacing 25 embryos in 17 embryo transfer cycles, yielded 6 pregnancies and 5 of them resulted in delivered or are ongoing. Limitations, reasons for caution We demonstrated that physiological rhPLCζ-AOA achieved an excelled fertilization and clinical outcomes comparing to the traditional ionomycin method. Although further observation is needed to confirm its safety, this approach represents a more physiological method capable of achieving fertilization, higher embryo cleavage and ultimately better pregnancy outcomes. Wider implications of the findings While ICSI has gained large popularity, poor fertilization may occasionally occur. The availability of an assisted oocyte activation that utilize a native compound capable of restoring fertilization capability of the male gamete is appealing and holds great promises. Trial registration number N/A

Effect of chemical activators after intracytoplasmic sperm injection (ICSI) on embryo development in alpacas

Low motility and low sperm concentration are characteristics of alpaca semen. Thus, the intracytoplasmic sperm injection (ICSI) technique represents an alternative to improve the reproductive capacity of the male. However, the effect of post-ICSI activation in alpaca is not yet known. The aim of the present study was to compare the effect of chemical activators on alpaca embryo development after ICSI. Alpaca ovaries were collected from a local slaughterhouse and transported to the laboratory. Category I, II and III oocytes were matured for 30 h at 38.5 °C. After ICSI, injected oocytes were randomly divided and activated as follows: i) 5 μM ionomycin for 5 min, ii) 7% ethanol for 4 min, iii) 5 μM ionomycin for 5 min, window period 3 h plus 7% ethanol for 4 min, iv) 5 μM ionomycin for 5 min, window period 3 h, a second ionomycin treatment for 5 min, followed by 1.9 mM 6-DMAP for 3 h, v) 10 mM SrCl2 for 3 h. Culture was carried out for 5 days in SOFaa at 38.5 °C. The cleavage rate was the lowest in the SrCl2 group, morula development was the lowest in the SrCl2 and without activation groups, and blastocyst stage was not different between groups (P<0.05). The rates with SrCl2 were lower in total embryos produced, whereas in transferable embryos they were lower with 2Io/6-DMAP and with SrCl2 (P<0.05). In conclusion, alpaca oocyte activation is more efficient with ionomycin and ethanol to produce transferable embryos.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Effect of follicle size on in vitro maturation in alpaca oocytes (Vicugna pacos) and the first ICSI in alpaca species

The development of reproductive biotechnologies is essential for the productive improvement of South American camelids. The present study was conducted to examinate the potential maturation of alpaca’s oocytes derived from small and large follicles, as well as perform intracytoplasmic sperm injection (ICSI) after in vitro maturation (IVM). Ovaries were collected from a slaughterhouse and transported at 10 °C for 22 h. Cumulus-oocyte complexes (COCs) were obtained by aspiration from large follicles (2–8 mm), while COCs from small follicles (<2 mm) were obtained by performing superficial sections on the ovary cortex. COCs of I and II categories were selected and subjected to IVM for 32–36 h at 38.5 °C. ICSI was performed according to standard protocols. IVF was performed as control for ICSI. After ICSI, oocytes were chemically activated by 5 min with 5 µM Ionomycin and 3 h with 6-DMAP. The results showed that yield of morphologically good-quality graded COCs was significantly higher in large follicles (2.1 ± 0.8) than from the small ones (1.2 ± 0.7). However, after IVM no significant differences were observed in the rate of metaphase II (18.1% vs 22.1%) and degenerated oocytes (48% vs 57%). In the following experiment, ICSI (n = 27) was performed and compared against IVF (n = 46). The 44% of chemically activated oocytes cleaved after ICSI vs 37% after IVF. DNA staining of the presumptive zygotes after 168 h of embryo culture showed that 11% of IVF and 19% of ICSI embryos were at pronuclear stage; then 7% of IVF and 12.5% of ICSI zygotes reached the 2-cell stage. Subsequently, 15% of IVF and none ICSI reached the 4–8 cell stage. Finally, 4% of IVF and 12.5% of ICSI were at morula stage after 7 days of culture. Thus, our findings indicate that larger follicles yield a greater amount of morphologically high-quality oocytes, but also oocytes from smaller follicles might reach similar IVM competence. In addition, this study represents preliminary development of IVF and ICSI embryos in alpaca; however, more research is needed to optimize IVP conditions in this species.

Low pH up-regulates interleukin-6 mRNA in L6-G8C5 rat skeletal muscle cells independent of pH sensing by SNAT2(SLC38A2) transporters.

Exercise is known to create a transient, but potent increase in skeletal muscle expression of potentially anti‐inflammatory myokine interleukin‐6 (IL‐6). This effect may be clinically important in managing chronic inflammatory states. It has previously been proposed that lactic acidosis following exercise promotes this IL‐6 up‐regulation, but the mechanism of this acidosis effect is unknown. Rat skeletal muscle cell line L6‐G8C5 has been used previously to model metabolic effects of acidosis, sensing low pH through the resulting inhibition of amino acid transporter SNAT2(SLC38A2). Use of ionophore ionomycin to model the rise in intracellular Ca2+ concentration occurring in contracting muscle strongly up‐regulates IL‐6 mRNA in L6‐G8C5 myotubes. This study used this model to test the hypothesis that low extracellular pH (7.1) enhances ionomycin‐induced IL‐6 mRNA up‐regulation by inhibiting SNAT2. Incubation of L6‐G8C5 myotubes for 6 h with 0.5 µM ionomycin at control pH (7.4) resulted in a 15‐fold increase in IL‐6 mRNA which was further enhanced (1.74‐fold) at pH 7.1. In contrast low pH had no significant effect on IL‐6 mRNA without ionomycin, nor on the IL‐6 mRNA increase that was induced by cyclic stretch. Even though pH 7.1 halved the transport activity of SNAT2, alternative methods of SNAT2 inhibition (JNK inhibitor SP600125; SNAT2 antagonist MeAIB; or SNAT2 silencing with siRNA) did not mimic the enhancing effect of low pH on IL‐6 mRNA. On the contrary, JNK inhibition blunted the effect of pH 7.1 with ionomycin, but had no effect at pH 7.4. It is concluded that low pH promotes Ca2+/ionomycin–induced up‐regulation of IL‐6 mRNA through a novel SNAT2‐independent JNK‐dependent pH‐sensing pathway not previously described in this skeletal muscle model.

Calcium signaling induces a partial EMT.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating invivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.

In vitro maturation of equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (ICSI) or parthenogenic activation

The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol.Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solutions (Experiment I), oocytes were exposed to vitrification media without cryopreservation. Of all the experimental groups evaluated, the best results were obtained for IVM oocytes exposed to EquiproVitKit media (IVM + TOX EquiVitKit), with a viability rate of 69.5%. In the Experiment II, oocytes, either freshly collected from the ovary or after in vitro maturation (IVM), were vitrified using either the EquiPro VitKit or an in-house medium containing 18% Ficoll, 40% ethylene glycol and 0.3 M sucrose. Oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate viability. In vitro matured oocytes vitrified using EquiproVitKit media (IVM + VIT EquiVitKit) had a cryosurvival rate of 63%. In the last part of the study (Experiment III), vitrified IVM oocytes were activated by 7.5 μM ionomycin in TCM-199 for 5 min TCM 199 (5 min) combined with 2 mM 6-DMAP in TCM-99 with 10% FBS (4.5 h) or in vitro fertilized using ICSI. Development of potential embryos after activation in TCM-199 medium, showed a cleavage rate was 10.2%, compared to 22.5% of oocytes cultured in G1/G2 medium. ICSI of vitrified IVM oocytes resulted in 20% embryo development to the 16-cell stage, compared to 33.3% in the control. The vitrification of oocytes after IVM by Rapid-I method is a good way to preserve genetic material in horses.

Development of prepubertal goat oocytes after their in vitro maturation and chemical activation.

Experiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus-oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification.

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7µM ionomycin in TCM 199 medium (5min) followed by 2mM 6-DMAP in TCM 199 supplemented with 10% FBS (3hr), then cultured and evaluated every 24hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.

Effect of vitrification of in vitro matured prepubertal goat oocytes on embryo development after parthenogenic activation and intracytoplasmic sperm injection

This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22–24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (Me2SO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2′7′ dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV− PI−), early apoptotic (AV+ PI−), dead non-apoptotic (AV− PI+) and necrotic (AV+ PI+). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.

111 Fertilizing ability of frozen and freeze-dried semen following intracytoplasmic sperm injection of invitro-matured sheep oocytes

Freeze-drying is a novel technique that permits the storage of semen at room temperature for long time periods, retaining their fertilizing capacity. The main objective of this work was to compare the fertilization ability of frozen-thawed (FT) and freeze-dried (FD) ram semen following intracytoplasmic sperm injection (ICSI) of invitro-matured (IVM) sheep oocytes. Oocytes were recovered by slicing the ovaries of slaughtered sheep. Selected cumulus-oocyte complexes (COCs) were IVM for 24h in tissue culture medium 199 (TCM-199) supplemented with 10% heat-treated oestrous sheep serum (ESS), 0.36mM pyruvate, FSH (1IUmL−1), and luteinising hormone (LH; 1IUmL−1) under mineral oil in a humidified atmosphere of 5% CO2, at 38.5°C. Semen was collected from fertile adult rams using an artificial vagina and processed for (1) freezing and thawing (Khalifa and Lymberopoulos, 2013 Cell Tissue Bank 14, 687-698; https://doi.org/10.1007/s10561-012-9357-6) or (2) freeze-drying and rehydration according to Arav et al. (2018 J. Assist. Reprod. Genet. 35, 1149-115; https://doi.org/10.1007/s10815-018-1145-1) protocols. For FD protocol, sperm samples were diluted in a sugar solution of trehalose and sorbitol (LyoB) and dehydrated for 24h. Later, the samples were rehydrated in a warming solution and diluted in TCM-199 before ICSI. After maturation, metaphase II (MII) oocytes with a polar body were injected with FT or FD sperm. Briefly, oocytes were transferred into groups of six in an ICSI dish containing 6-µL drops of holding medium (TCM-199 + 5% fetal bovine serum) and 3-µL drops of PVP for the sperm samples. Injection was carried out with an inverted microscope (Olympus IX73) connected to a micromanipulation system (Narishige) using ICSI pipettes with 7-µm internal diameter. Within 1h, ICSI oocytes were activated with 5 µM ionomycin for 4min and invitro cultured in modified synthetic oviductal fluid medium (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817; https://doi.org/10.1071/RD11023). After 17-21h, injected oocytes were fixed and stained in a solution of ethanol Hoechst 33342 and classified as FPN (one female pronucleus and one condensed sperm head), MPN (one male pronucleus and one MII), 2PN (two pronuclei, male and female), 3PN (three or more pronuclei), and NPN (no pronuclei). Data were analysed using analysis of variance (two-way ANOVA) followed by Tukey post hoc test with SAS software, version 9.4. The ICSI-FD group had a higher number of NPN and a lower number of 2PN than did the ICSI-FT group (P&amp;lt;0.05). We think that more technical advances in the FD process as well as the rehydration procedure are necessary to improve the application of FD ovine semen for invitro fertilization by ICSI in sheep, but in any case these results have showed that FD could be a useful tool for the future of invitro embryo production. Table 1.Pronuclear formation at 17-21h post-injection1 Treatment n FPN MPN 2PN 3PN NPN FT 71 9.66±4.12 4.26±1.48 48.13±2.79a 5.97±4.16 31.98±6.75a FD 65 6.16±2.26 1.39±1.39 20.15±4.14b 10.57±6.59 61.73±6.89b a,bValues in the same column with different superscript letters differ significantly (P&amp;lt;0.05). 1Data are presented as mean±s.e.m. FPN=female pronucleus, MPN=one male pronucleus and one metaphase II oocyte, 2PN=two pronuclei, male and female, 3PN=three or more pronuclei, NPN=no pronuclei. Funding was provided by Spanish MINECO Grant AGL2017-85837-R, Spanish MECD pre-doctoral grant FPU15/00773, and Spanish MECD mobility grant EST18/00472 to Irene Menéndez Blanco.

Direct Visualization of Circulating Neutrophil Extracellular Traps Using Cell Fluorescence during Human Septic Shock-Induced Disseminated Intravascular Coagulation

Septic shock is the most severe form of infection, defined as a subset of sepsis in which circulatory, cellular and metabolic abnormalities are profound and responsible for multiple organ failure and a high mortality-rate. Septic shock is characterized by a broad coagulation activation that can lead to uncontrolled thrombin and fibrin generation, which may evolve to disseminated intravascular coagulation (DIC). DIC increases the risk of death, thus representing a therapeutic target of interest. However, the pathophysiological mechanisms of DIC are not fully understood. Polymorphonuclear neutrophils (PMNs) have recently been identified as potential players of the response to infection by releasing their content, including DNA, histones and granules enzymes. These structures, called neutrophils extracellular traps (NETs), form a large net-like structure in which pathogens get trapped. NETs capture invading pathogens, but also represent a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. In this study, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC using a simple and robust method. Blood samples were obtained from healthy human volunteers (n=3), patients with septic shock without DIC (n=3) and with DIC (n=3). PMNs were immediately purified immediately after sampling using negative immune-magnetic sorting method. 5x104 purified PMNs were subsequently spotted on microscope slides using cytocentrifugation at 35g for 5 minutes, thus preserving cell integrity and morphology. Positive controls for NET formation were obtained using PMNs isolated from healthy volunteers that were stimulated in-vitro with 4 μM ionomycin. PMNs were stained with mouse anti-human FITC anti-myeloperoxidase (MPO) antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DAPI stained DNA fibers associated to MPO detected by immunofluorescence microscopy. The degree of NETs formation was blindly quantified into 5 categories according to the proportion of neutrophils forming NETs per microscopic field: absence (0% of neutrophils forming NETs), scarce (1-25%), moderate (26% to 50%), high (50% to 75%) or very high degree of NETS formation (76% to 100%). PMNs from healthy donors stimulated by ionomycin released NETs, evidenced as double-stained DAPI-positive chromatin structures decorated with MPO in the extracellular space using fluorescence microscopy. NET structures similar to those observed after ionomycin stimulation were unambiguously visualized in PMNs from septic shock patients with DIC, but neither in PMNs from septic shock patients without DIC, nor in unstimulated PMNs from healthy donors. DIC patients were stratified into "high" or "very high" degree of NETs formation groups (&amp;gt;75 % of PMNs displaying DNA release), not different from ionomycin-stimulated PMNs (50 to 75%). Conversely, PMNs from patients devoid of DIC as well as unstimulated normal PMNs from healthy volunteers were stratified into "scarce" or "absence" of PMNs forming NET categories. Blind review of May-Giemsa-Grunwald (MGG) stained slides showed the correlation of the detection of NETs with nuclear decondensation and vacuolation of the cytoplasm of PMNs, features that are associated with neutrophil activation. Accordingly, neutrophils' side-fluorescence (NEUT-SFL), a Sysmex™ CBC analyzers parameter previously associated to NETosis (Stiel et al., 2016, Delabranche et al., 2017) was increased during septic shock induced DIC as well as following in-vitro ionomycin stimulation of normal PMNs. In this study, we showed for the first time direct evidence of circulating NETs in peripheral blood of patients with septic shock-induced DIC using immunofluorescence. These NETs are undistinguishable from NETs features observed following in-vitro ionomycin stimulation of normal PMNs. NETs images were furthermore associated with modifications of PMN morphology that could be detected using MGG staining. This observation may be of great interest in order to stratify patients eligible for future treatment protocols with molecules targeting NETs. Figure Disclosures Meziani: STAGO: Research Funding.

Calcium-calmodulin gating of a pH-insensitive isoform of connexin43 gap junctions.

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (gj) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 μM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 gj to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 gj, while 100 μM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (Vj) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 gj, but also noted the abolishment of the previously reported facilitated recovery of gj from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the Vj-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

158 Predicting embryo development success with physical parameters of oocytes

There is a continual search for reliable, noninvasive methods of selecting viable oocytes and embryos. Previous studies have indicated that the physical size of oocytes may reflect their developmental potential. The objective of this study was to observe the correlation between an oocyte’s diameter [including zona pellucida (ZP), cell area, and ZP thickness] and its ability to develop. Bovine cumulus-oocyte complexes were collected from abattoir-derived ovaries and incubated for 24h in TCM-199-based maturation medium. The cumulus-oocyte complexes were denuded by vortexing with hyaluronidase, and mature oocytes were selected based on presence of a visible polar body. Selected oocytes were artificially activated by incubating in 5 μM ionomycin for 5min followed by incubation in 2mM 6-DMAP for 3h (n=723). After activation, oocytes were placed in individual 5-μL culture droplets under oil and photographed using an inverted scope with digital camera. Oocytes were then group cultured in 50-μL droplets in a polyester micromesh for identification of individual oocytes. Development to the blastocyst stage was noted on Days 7 and 8 of culture. ImageJ was used to measure diameter, area, and ZP thickness from images. A logistic regression using the lme4 package in R was run with Day 7 or 8 blastocyst development as the response; diameter, area, and ZP thickness as predictors; and replicate and culture droplet as random effects. The residual estimates of area and ZP thickness from diameter were used to account for correlation between predictors. Only significant interactions were kept in the model. The ZP thickness ranged from 6.1 to 17.7μm with a mean of 12μm. There was a significant correlation between diameter and ZP thickness (P&amp;lt;0.01, R2=0.12) with a correlation coefficient of 0.35. Oocyte diameter had a significant effect on subsequent blastocyst development on Day 7 (P&amp;lt;0.01) and Day 8 (P&amp;lt;0.01), with larger oocytes more likely to develop on both days. Oocytes were also grouped into quantiles by diameter. Larger groups were more likely to develop to the blastocyst stage on Day 7 (P&amp;lt;0.001) and Day 8 (P&amp;lt;0.001). Blastocyst rate on Day 8 for oocytes with diameters &amp;lt;149.5μm was 24.2%, whereas blastocyst rate on Day 8 for those with diameters=159μm was 41.2%. In addition, ZP thickness also had an effect: oocytes with thinner ZP were more likely to develop to the blastocyst stage on Day 7 (P&amp;lt;0.001) and Day 8 (P&amp;lt;0.001). Blastocyst rate for oocytes with a ZP thickness &amp;lt;11μm was 37%, whereas the rate for those with a ZP thickness of 12.9μm was 27.6%. Area did not have an effect on blastocyst formation on Day 7 or 8 (P=0.21). Here we have demonstrated that the oocyte diameter, including the ZP and ZP thickness, affects its probability of development. Larger oocytes and those with thinner ZP are more likely to develop to the blastocyst stage. Differences in the size of the perivitelline space could explain why diameter had a significant effect on development and area did not. Further studies will focus on determining the relationship between these physical parameters of oocytes and embryo quality.

Culture conditions affect Ca2+ release in artificially activated mouse and human oocytes.

Inconsistent fertilisation and pregnancy rates have been reported by different laboratories after application of ionomycin as a clinical method of assisted oocyte activation (AOA) to overcome fertilisation failure. Using both mouse and human oocytes, in the present study we investigated the effects of ionomycin and Ca2+ concentrations on the pattern of Ca2+ release and embryonic developmental potential. In the mouse, application of 5μM ionomycin in potassium simplex optimisation medium (KSOM) or 10µM ionomycin in Ca2+-free KSOM significantly reduced the Ca2+ flux and resulted in failure of blastocyst formation compared with 10μM ionomycin in KSOM. Increasing the Ca2+ concentration up to three- or sixfold did not benefit mouse embryonic developmental potential. Similarly, 10μM ionomycin-induced rise in Ca2+ in human oocytes increased with increasing total calcium concentrations in the commercial medium. Remarkably, we observed significantly reduced mouse embryo development when performing AOA over a period of 10min in Quinn's AdvantageTM Fertilisation medium (Cooper Surgical) and IVFTM medium (Vitrolife) compared with Sydney IVF COOK cleavage medium (Cook Ireland), using the same sequential culture system from the post-activation stage to blastocyst formation stage in different AOA groups. In conclusion, concentrations of both ionomycin and Ca2+ in culture media used during AOA can have significant effects on Ca2+ release and further embryonic developmental potential.

Effects of different activation protocols on cleavage rate and blastocyst production of caprine oocytes.

The present study was undertaken to assess the effect of different chemical activators along with 6-DMAP on in vitro matured caprine oocytes. From 4332 ovaries, 14235 cumulus oocyte complexes (COCs) were collected which were matured in TCM-199 medium containing follicle stimulating hormone (FSH) (5 µg/ml), Leutinizing hormone (LH) (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% fetal bovine serum, 10% follicular fluid and 3 mg/ml bovine serum albumin (BSA) at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. In group 1 (control), 3117 in vitro matured oocytes were co incubated with sperms for 18 h in ferttalp medium. In group 2, 3563 in vitro matured oocytes were activated with 7% ethanol for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 3, 3109 in vitro matured oocytes were activated with 5 μM ionomycin for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 4, 3455 in vitro matured oocytes were activated with 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Oocytes were cultured in 50 µL drops of research vitro cleave (RVCL) medium for embryo development. The cleavage rate, morula and blastocyst production in group 1, 2, 3 and 4 were 26.07 ± 2.37%, 14.91 ± 2.91 & 1.45 ± 0.71%, 49.57 ± 3.79%, 20.07 ± 2.38% & 5.29 ± 1.42%, 50.18 ± 3.59%, 15.26 ± 2.87% & 1.85 ± 0.72% and 80.26 ± 2.30%, 35.33 ± 2.67 & 7.10 ± 0.89%, respectively. These results indicated that the activation of in vitro matured oocytes by 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h is most favorable for parthenogenetic caprine embryos production.

34 Recent Advances in Cloning by Somatic Cell Nuclear Transfer in Camelids

In addition to its application for production of elite males, racing champions, animals with the highest potential for milk production, and the prized beauty camels, somatic cell nuclear transfer (SCNT) can be utilised for the conservation of endangered wild Bactrian camels and vicunas by using the technique of interspecies SCNT (iSCNT). In the present study, embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus), a llama (Llama glama), and a dromedary camel (Camelus dromedarius) as donor karyoplasts and dromedary oocytes as recipient cytoplasts to evaluate in vitro and in vivo developmental potential of these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated oocytes. The fibroblast cell and recipient cytoplasm were fused by 2 DC pulses of 100V for 15 µs each. Reconstructs were activated 1 to 1.5 h post-fusion with 5 µM ionomycin, followed by exposure to 6-DMAP for 4 h. The activated oocytes were then cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. A lower blastocyst production rate was observed when llama fibroblast cells were used as karyoplast (13.3 ± 3.3) compared with those obtained from the embryos reconstructed with Bactrian (34.4 ± 3.9) and dromedary (32.2 ± 6.2) fibroblast cells; however, no difference was observed in their cell numbers. Out of 26 and 20 blastocysts from reconstructs with Bactrian and dromedary fibroblast cells, transferred to 23 and 12 synchronized dromedary recipients, 3 and 2 pregnancies were achieved at Day 60, respectively. One pregnancy from each group reached term and both (a Bactrian and a dromedary) calves were delivered normally after completing the gestation period. We demonstrated birth of a Bactrian calf conceived from the reconstructed embryo by iSCNT using the somatic cell from a Bactrian camel and enucleated dromedary oocyte. The present study also demonstrated that dromedary camel can be used as a surrogate to carry these pregnancies to term (Wani et al. 2017 PLoS One 12, e0177800; https://doi.org/10.1371/journal.pone.0177800). This study has opened doors for enhanced multiplication and preservation of the wild Bactrian camels, which are threatened with extinction, being the eighth most endangered large mammal on the planet.

29 Co-Incubation of Equine Cloned Embryos with Sialic Acid: Effect on Pregnancy Rate

In vitro-produced equine embryos have certain morphological characteristics that differ from embryos produced in vivo. One of them is the absence or inadequate formation of the embryo capsule. This capsule is composed of mucin-like glycoproteins produced by the trophectoderm with high proportion of sialic acid, which confers anti-adhesive properties. This characteristic is necessary for the intrauterine embryo migration process to occur, which is vital and fundamental for pregnancy recognition. For this reason, inadequate formation of the glycoprotein capsule could result in a lower pregnancy rate. In this study, we aimed to evaluate the effect of cloned equine embryo co-incubation with sialic acid on blastocyst and pregnancy rates. To achieve this, equine oocytes obtained from slaughterhouse ovaries were matured in TCM-199 HEPES medium with 2% fetal bovine serum, 2% antibiotics, and 1 μg mL−1 FSH, incubated at 39°C for 24 h. Matured oocytes were denuded with pronase, enucleated, and fused to donor bone marrow mesenchymal cells according to Olivera et al. (2016 PLoS One 11, e0164049, 10.1371/journal.pone.0164049). Chemical activation was induced using 8.7 μM ionomycin for 4 min and embryos were incubated with 1 mM 6-DMAP and 5 mg mL−1 cycloheximide for 4 h. Afterwards embryos were cultured in microwells for 8 days in DMEM-F12 medium. On Day 6, cloned equine embryos were exposed to 5 μM sialic acid for 48 h (SA group). On Day 8, blastocysts were transferred to recipient mares 5 days post-ovulation and pregnancy was confirmed 15 days post-transfer by transrectal ultrasound. Embryo clones generated without sialic acid exposure were used as a control (C) group. Fisher test was used to analyse both blastocyst and pregnancy rates. Blastocyst rates were 14% (46/328) and 15% (62/413) and pregnancy rates were 30.4% (7/23) and 19.4% (6/31) for SA and C groups, respectively. No statistical differences were observed between groups for the analysed parameters, even though pregnancy rates tended to be higher in the SA group. This effect could be a consequence of higher concentrations of the glycoprotein involved in the formation of the embryo capsule.

35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P &lt; 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT. Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells

128 NEURONATIN TRANSCRIPT ANALYSES AS MARKER TO CONFIRM SUCCESSFUL FERTILIZATION IN BOVINE BLASTOCYSTS

In humans, intracytoplasmic sperm injection (ICSI) has become widely applied to overcome infertility. In addition to its clinical usefulness, ICSI is also a valuable research tool for studying fundamental aspects of gamete interaction during fertilization. Oocyte activation is a prerequisite for decondensation of the sperm head and formation of the male pronucleus regardless of whether the spermatozoon enters the oocyte by injection or by fertilization. In several species, the injection procedure itself is sufficient to activate the oocyte. However, the bovine oocyte is rarely activated by the mechanical stimulation of injection alone. Therefore, additional stimuli are required during ICSI, which may also initiate parthenogenetic development. Because blastocyst origin, either via IVF or after parthenogenetic activation, could not be distinguished at the morphological level via microscopic evaluation, the paternal genome needs to be determined, employing a molecular approach. Therefore, the aim of this study was to validate the expression of neuronatin (NNAT) mRNA, a paternally expressed gene, via RT-PCR, to verify whether bovine blastocysts were generated by fertilization or parthenogenesis. Bovine cumulus-oocyte-complexes were recovered from abattoir-derived ovaries by the slicing method. After 24 h of maturation, fertilization was achieved using a standard protocol. Presumptive zygotes were cultured in SOFaa for a period of 8 days. To generate parthenogenetic blastocysts, oocytes were activated, employing the following protocol. After 24 h of maturation, the oocytes were exposed to 5 µM ionomycin for 5 min and were then incubated in 1.9 mM 6-dimethylaminopurine for 4 h. Afterwards, treated oocytes were cultured in SOFaa for 8 days. Blastocysts from both experimental groups (IVF-derived and parthenogenetic) were stored at −80° until analysis. RT-PCR was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–510) for 6 embryos stemming from the IVF-group and 3 embryos out of the chemical-activated group. The NNAT mRNA was expressed in 5 blastocysts stemming from the IVF group; one blastocyst did not show NNAT mRNA expression, suggesting that it was derived from parthenogenesis. Conversely, NNAT mRNA could not be detected in all blastocysts derived from chemical activation, indicating that only the maternal genome was present. These results clearly demonstrate that NNAT mRNA expression is a reliable method to prove the paternal participation in early bovine embryo development.