Calcium Ionophore Research Articles

MiR-6918-5p prevents renal tubular cell apoptosis by targeting MBD2 in ischemia/reperfusion-induced AKI

AimsAlthough previous studies reported that miRNAs are involved in the progression of acute kidney injury (AKI), their exact function and mechanism in ischemic AKI remains largely unknown. This study aims to define the role of miR-6918-5p in ischemia-reperfusion AKI. Materials and methodsThe renal arteries of C57BL/6J mice were clamped to establish a model of ischemia-reperfusion renal injury. BUMPT cells were added with Antimycin A and calcium ionophore to establish a model of ATP depletion in vitro. Cell apoptosis was detected by CCK8, flow cytometry and western blot, while HE staining and TUNEL staining were used to assess the degree of kidney damage. Key findingsWe suppressed mmu_miR-6918-5p by ischemic injury in vitro and in vivo. We found that ischemia-reperfusion (I/R)-induced renal tubular cell apoptosis and the expression of cleaved caspase3 were enhanced by the inhibitor of mmu_miR-6918-5p; this effect was attenuated by an mmu_miR-6918-5p mimic. Mechanistically, mmu_miR-6918-5p binds to the 3′ UTR region of MBD2 and represses its expression. The mmu_miR-6918-5p mimic alleviated the ischemic AKI by targeting MBD2. Conversely, the inhibitor of mmu_miR-6918-5p enhanced the ischemic AKI; this was diminished by MBD2-KO. SignificanceMmu_miR-6918-5p protected against the development of ischemic AKI by targeting MBD2.

Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

High-performance amperometric determination of nitric oxide released by endothelial cells using flow injection analysis

An amperometric flow injection analysis (FIA) method based on an optimized carbon paste electrode (CPE) for the determination of nitric oxide (NO) production from endothelial cells (ECs) was developed. The CPE was modified by Nafion, gold nanoparticles (AuNPs) and graphene nanoribbons (GNRs) via drop casting method. The proposed sensor exhibited excellent sensitivity and selectivity to the oxidation of NO with a linear correlation of 0.02 - 22.4 μM and a detection limit of 6 nM. The developed method is applied for real-time measurements of NO released from endothelial cells by stimulation of endothelial NO synthase (eNOS). Induction of NO synthesis by eNOS was successfully initiated by the calcium ionophore A23187, acetylcholine (ACh), or L-arginine. Furthermore, a reduction of eNOS activity could be observed after the addition of a NOS inhibitor, resulting in a decrease in NO signaling. Targeting eNOS could be of considerable value with regard to preventing or treating the development of cardiovascular diseases. The sensor’s ease of fabrication and applicability for cell measurements enables the device to be used in pharmacological research and drug discovery.

Pathogenic variant in ACTL7A causes severe teratozoospermia characterized by bubble-shaped acrosomes and male infertility.

Teratozoospermia is a common factor associated with male infertility. However, teratozoospermia characterized by bubble-shaped acrosomes (BSAs) has not yet been identified in men and the causative genes are unknown. The present study is of a patient with severe teratozoospermia characterized by BSA and carrying a variant (c.1204G>A, p.Gly402Ser) of actin-like 7A (ACTL7A). For further verification, we generated an Actl7a-mutated mouse model (p.Gly407Ser) carrying an equivalent variant to that in the patient. We found that homozygous Actl7a-mutated (Actl7aMut/Mut) male mice were sterile, and all their sperm showed acrosomal abnormalities. We detected by transmission electron microscopy that during acrosomal biogenesis, the acrosome detaches from the nuclear membrane in Actl7aMut/Mut mice. Furthermore, mutant ACTL7A failed to attach to the acroplaxome and was discharged by cytoplasmic droplets, which led to the absence of ACTL7A in epididymal spermatozoa in mice. The mutant sperm failed to activate the oocyte, and sperm-borne oocyte activation factor phospholipase C zeta (PLCζ) discharge accompanied by ACTL7A was observed, leading to total fertilization failure (TFF). Immunoprecipitation followed by liquid chromatography-mass spectrometry showed that several differentially expressed proteins participate in acrosome assembly and actin filament organization. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF in the couple with an ACTL7A pathogenic variant. Our study defined a novel phenotype of an acrosomal abnormality characterized by BSA, revealed the underlying mechanism of a pathogenic variant in ACTL7A and provided a genetic marker and potential therapeutic option for male infertility.

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa

Research questionDoes sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process? DesignHuman spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge. ResultsSIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca2+ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237. ConclusionYK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.

Functional significance of mouse seminal vesicle sulfhydryl oxidase on sperm capacitation in vitro.

During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.

P-139 The incidence of monozygotic twins after the usage of calcium ionophore or after trophectoderm biopsy in ART treatment

Abstract Study question To investigate whether the usage of calcium ionophore or trophectoderm biopsy affect the occurrence of monozygotic twins (MZT) in assisted reproductive treatment (ART) treatment. Summary answer Despite the increase was not statistically significant, higher percentage of MZT were observed in both group with calcium ionophore usage and trophectoderm biopsy. What is known already The incidence of monozygotic twins (MZT) in natural conception is approximately 0.4% worldwide but it is estimated that there is at least a 2-fold rise in the incidence of MZT after assisted reproductive technology compared with natural conception, although the reason is still under debate. The effectiveness of calcium ionophore in assisting oocytes activation and fertilisation has been widely studied. Most studies reported no impact upon embryo viability and reproductive potential after trophectoderm biopsy. However, the impact of calcium ionophore or trophectoderm biopsy on subsequent in vivo development of the blatocyst remains unclear. Study design, size, duration This is a retrospective observational cohort study of frozen embryos transfers (FET) that have achieved clinical pregnancy between January 2019 and October 2021. In this study, 709 FET cycles were included and analysed based on i) with or without calcium ionophore treatment after ICSI/IVF on oocytes retrieval day; ii) with or without trophectoderm biopsy on day 5 or 6. The results were analysed to identify possible risk factors affecting the prevalence of monozygotic twins. Participants/materials, setting, methods The cases with positive beta-hCG test were followed-up and analysed for the occurrence of MZT. The diagnosis of MZT was established by transvaginal ultrasound performed at 4-6 weeks after the transfer. The visualization of uteri containing more than one fetus, either in one sac (monochorionic monoamniotic) or in two sacs separated by a septum (monochorionic diamniotic) is suggestive of MZT. Data were analysed by Fisher’s exact test and Odd Ratio. Main results and the role of chance Data comprising a total of 709 FET cycles in a single clinical setting were analysed. Among 449 cases of clinical pregnancies, 8 cases of MZT occurred. A higher percentage of MZT (1.78%) after blastocyst transfer in ART treatment was observed as compared to natural conception (0.04%). There was a higher incidence of MZT in trophectoderm biopsy group as compared with non-biopsy group (2.3% vs 1.45% respectively, odds ratio [OR]= 1.59, 95% CI 0.39–6.46). However, there was no statistically significant (P-value=0.556, CI = 95%). The incidence of MZT following the calcium ionophore usage was higher compared with the group without calcium ionophore usage (2.33% versus 1.72%, OR = 1.36, 95% CI 0.16–11.3). There was no significantly higher risk of MZT with calcium ionophore versus non-calcium ionophore cycles (P-value= 0.493, CI = 95%). Limitations, reasons for caution The sample examined in this study is limited in size thus more studies are needed to achieve satisfactory statistical power. MZT diagnosed with ultrasound is difficult to differentiate from dichorionic monozygotic twins. The definitive diagnosis of monozygosity is only possible by DNA analysis of all the twins born. Wider implications of the findings These findings can draw attention to study the effect of calcium ionophore on embryo development and the mechanism behind MZT. Based on the findings, the patients undergoing ART treatment should be informed with the risk associated with higher incidence of MZT. Trial registration number NA

O-215 Embryo developmental competence of de novo male gametes in a novel three-dimensional culture

Abstract Study question Are de novo gametes generated from mouse embryonic stem cells (mESCs) in a novel three-dimensional (3D) culture system capable of supporting full preimplantation development? Summary answer A novel 3D culture system yielded de novo male gametes capable of fertilizing an oocyte and supporting full preimplantation development. What is known already In regenerative medicine, several 3D culture systems have been shown to be capable of producing functional tissue implants. In reproductive biology, recent studies have reported preliminary success in generating functional de novo gametes through soft-agar culture and testicular organoids, in the mouse model. However, a heterologous transplantation technique is required to obtain functional gametes. Study design, size, duration mESCs were first cultured on a gelatin-coated 6-well plate with fibroblasts in monolayer and later spherified using sodium alginate. Spheres were submerged in specifically designed conditioned media to encourage differentiation of the mESCs into germ-like cells. Over the course of differentiation, cells were assessed for germ cell differentiation biomarkers. Considering that normal spermatogenesis occurs in 30 days, utilization of the de novo gametes was planned for days 15, 22, 29, and 36. Participants/materials, setting, methods mESCs were differentiated by submerging the spheres in EpiLC medium containing Activin A, bFGF, and KSR for 3 days followed by PGCLC medium containing BMP4, LIF, SCF, and EGF for up to 36 days. Differentiation was assessed for markers DAZL (spermatogonium), VASA (spermatocyte), BOULE (post-meiotic stage), and acrosin (spermatid). Differentiated cells were then injected into oocytes and activated by calcium ionophore. Embryo development was monitored in a time-lapse incubator. Main results and the role of chance Expression of DAZL in 20% and VASA in 15% of the cells at day 3 demonstrated progression into spermatogenesis. On day 10, DAZL and VASA were assessed again, revealing increases to 45% and 18%. A small proportion of cells expressed post-meiotic biomarkers, BOULE (1%) and acrosin (2%). On day 15, VASA expression plateaued at 17%, BOULE expression peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased slightly to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, acrosin expression remained stable at 7%, and BOULE expression dropped to 2%. On day 36, VASA was still expressed at 13%, and few cells expressed acrosin (1%); there was complete loss of BOULE expression. Our mouse ICSI control achieved 89.2% fertilization and 77.8% blastocyst rates. De novo gametes were injected into oocytes on days 15, 22, 29, and 36, achieving fertilization rates of 35.0%, 61.1%, 81.8%, and 75.0%, respectively. Correspondently, hatching blastocysts were obtained at rates of 5.0%, 16.7%, 36.4%, and 8.3%, respectively. Limitations, reasons for caution Despite the ability to fertilize normally, the blastulation rate remained suboptimal. Most importantly, the ability to generate live offspring still needs to be documented. Wider implications of the findings Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allo-/xeno-geneic transplantation. If reproducibility and the ability to obtain healthy offspring are confirmed, this method may represent a tool for achieving neogametogensis in mammals. Trial registration number N/A

O-020 Sperm phospholipase C zeta levels measured by flow cytometry are associated with sperm parameters

Abstract Study question Are the levels of phospholipase C zeta (PLCζ) in sperm associated with sperm parameters? Summary answer Yes, PLCζ levels are correlated with motility, vitality, concentration and morphology and decreased in samples with anormal sperm parameters. What is known already Sperm phospholipase C Zeta (PLCζ) is a protein mainly located in the sperm head and its major role is the induction of the Ca2+ oscillations necessary for oocyte activation following fertilization. During fertilization, PLCζ is released into the ovum cytoplasm and induces Ca2+ oscillations via the inositol 1,4,5-triphosphate signaling pathway. PLCζ deficiency have been shown to be associated with low or total failed fertilization after intracytoplasmic sperm injection. In such cases, artificial oocyte activation (AOA) using calcium ionophore has been shown to increase fertilization rates. Thus, finding easy method to quantify PLCζ in a clinical setting is crucial. Study design, size, duration Between May 2021 and September 2021, 92 patients already doing a spermogram gave their inform consent to donate their unused sperm sample to research. They were split into two groups based on the normality of the spermogram (following World Health Organization 2010 criteria), normal sperm parameters (NPS, n = 24) and abnormal sperm parameters (ASP, n = 68). Levels of PLCζ were also determined in a subgroup of patient before and after the passage in a microfluidic chamber (ZyMōt™)(n = 27). Participants/materials, setting, methods Age, volume, pH, concentration, total sperm count, motility, progressive motility, vitality, morphology, DNA fragmentation, chromatin compaction, level of PLCζ and fluorescence intensity were compared between the two groups and correlations were made to better understand the association between PLCζ levels and sperm parameters. PLCζ levels were assessed by flow cytometry after fixation and permeabilization of the cells, primary antibody (rabbit anti-human PLCζ) and secondary antibody (goat anti-rabbit coupled with the fluorochrome FITC). Main results and the role of chance Significantly lower PLCζ levels were observed in ASP patients compared to NSP patients (respectively 45.4% vs 56.9%, p = 0.0038). Positive correlations were also observed between PLCζ levels and sperm concentration, total sperm count, progressive motility, total motility, and sperm vitality (p<0.001). ROC analysis revealed that a PLCζ levels cut-off of 64.4% could differentiate NSP from ASP patients with 83.8% sensitivity, 54.17% specificity, 54.2% PPV and 83.8% NPV (Area under the curve: 0.6829; p<0.0079). Moreover, higher PLCζ levels were found after sperm migration using a ZyMōt™ device (before 45.96 vs after 60.15, p<0.001). This method development also demonstrated the ability to detect low PLCζ levels in semen samples and those patients will be directed to artificial oocyte activation using a calcium ionophore to increase their chance of success. Limitations, reasons for caution Since there is a correlation between PLCζ levels and motility/vitality, it is expected that a sample treatment with a microfluidic chamber will increased the percentage of PLCζ. In addition, PLCζ is implicated in oocyte activation, so the real cut-off point should be determined with in-vitro fertilization (IVF) outcomes. Wider implications of the findings Demonstrating the association between PLCζ levels and sperm parameters established the relevance of using a microfluidic chamber for a better sperm quality for IVF or intracytoplasmic sperm injection (ICSI). Trial registration number Not applicable

P-797 Benefit of Replicating Mammalian Gametes

Abstract Study question Can haploid parthenogenetic and androgenetic embryos generate haploid embryonic stem cells (haESCs) and function as respective oocytes and spermatozoa? Summary answer Haploid embryos generated blastocysts capable of yielding haESCs. Haploid blastomeres of both sexes served as multiple copies of gamete substitutes. What is known already Although creating diploid digyneic or dispermic embryos through nuclear transfer is possible, the conceptuses rarely achieve normal preimplantation development due to unbalanced epigenomes, demonstrating the requirement of both parental epigenomes in the course of embryogenesis. Haploid parthenogenetic embryos can be achieved by inducing calcium oscillations of a metaphase II oocyte, while the paternal counterpart can be generated through the insemination of enucleated oocytes. These embryos may serve to generate haESCs for heterozygosity identification and can be used for reproductive purposes in animal models. Study design, size, duration To generate parthenogenetic embryos, metaphase II oocytes from B6D2F1 mice were artificially activated. Androgenetic embryos were generated from the insemination of enucleated oocytes. Haploid embryos were cultured to blastocysts and seeded on feeder cells (MEF) to derive haESCs. Haploid blastomeres from 8-cell embryos of either sex were isolated and used as artificial gametes; artificial oocytes were generated by grafting a parthenogenetic blastomere to an enucleated recipient oocyte. Androgenetic blastomeres were used as male gametes. Participants/materials, setting, methods Parthenogenetic embryos were generated by exposing metaphase II oocytes to calcium ionophore. Another cohort of B6D2F1 oocytes were enucleated and underwent piezo-ICSI to generate androgenetic embryos. Sendai-virus–mediated nuclear transfer was performed using parthenogenetic blastomeres as donor nucleuses to generate artificial oocytes and subsequently fertilized. Androgenetic blastomeres were used as sperm substitutes by fusion with an intact oocyte to generate zygotes. Control embryos were generated by piezo-actuated ICSI. Developmental characteristics were recorded by time-lapse microscopy. Main results and the role of chance A total of 129 oocytes were parthenogenetically activated by calcium ionophore, and 119 developed a female pronucleus. Androgenetic embryos were generated from the insemination of 375 ooplasts; 317 developed a male pronucleus. The monopronuclear appearance of haploid embryos of both sexes was comparable to the fertilization of control at 90.7%. Haploid embryo development for both sexes up to the 4-cell stage matched the control. However, from the 8-cell stage onward, parthenogenetic and androgenetic embryos cleaved at a lower rate compared to the control at 76.4%, 59.3% and 92.7% (P < 0.0001), respectively. The haploid genome affected the rate of compaction at 73.6%, 55.3%, and 89.2% (P<0.0001), respectively, and the rate of blastulation at 27.8%, 11.2%, and 80.8% (P<0.00001) respectively. Androgenetic embryos showed delayed cleavage from the 4-cell stage onward, while the parthenogenetic counterparts had similar morphokinetics to the control. A total of 20 parthenogenetic and 18 androgenetic blastocysts were plated on MEF, yielding 5 parthenogenetic and 3 androgenetic haESC lines. Both haESCs yielded beating cardiomyocytes on day 10 of differentiation while maintaining haploidy. The utilization of haploid pseudo blastomeres as gametes yielded 93 reconstructed biparental embryos that resulted in 71 blastocysts (76.3%). Time-lapse morphokinetics were comparable to the control. Limitations, reasons for caution Haploid embryos developed to blastocysts at a remarkably lower rate than the control, particularly the androgenotes; however, they can yield pluripotent haESCs capable of differentiation in the mesoderm and subsequent cardiogenesis prone to self-diploidization. Once this system is replicated in humans, the role of the centrosome needs to be investigated. Wider implications of the findings Our study has demonstrated the feasibility of propagating both male and female genomes from single gametes for reproductive applications. Indeed, once a stable culture of haESCs is achieved, genotyped pluripotent haESCs can be used to help identify heterozygosity for inherited disorders. Trial registration number N/A

P-798 Utilization of Haploid Androgenetic Blastomeres to Characterize Male Genomic Inheritance and as a Male Gamete Substitute

Abstract Study question Can sperm genomes be replicated by generating androgenetic embryos, and can resulting haploid blastomeres be used as a substitute for male gametes with known genotype? Summary answer Once utilized as male gametes, haploid androgenetic blastomeres can be used for gamete genotyping and can sustain embryo development to live offspring. What is known already Heterozygosity in human spermatozoa may represent a risk to offspring health. This has led to attempts to replicate the gamete genome by generating haploid embryonic stem cells (haESCs) that can be used to typify heterozygosity or as gamete substitutes. Although haESC lines are unstable with a tendency to self-diploidize, utilizing haploid androgenetic cells in the early embryonic stages, such as the blastomere stage, can provide multiple copies of genetically functional pseudo-gametes. Study design, size, duration Haploid androgenetic embryos were generated using spermatozoa from B6 heterozygous mice (B6-EGFP x B6D2F1) and cultured until the 8-cell stage. Individual haploid blastomeres with green fluorescent protein (GFP) expression were fused with activated recipient oocytes. Resulting conceptuses were cultured in a time-lapse system to monitor each stage of preimplantation development. Blastocysts with GFP expression were transferred to pseudo-pregnant surrogates. Pregnancy and offspring reproductive health were evaluated. Piezo-actuated ICSI was performed on unmanipulated oocytes as control. Participants/materials, setting, methods To generate haploid androgenetic embryos, metaphase II oocytes of B6D2F1 mice were treated with cytochalasin B, enucleated under Oosight®, and inseminated by piezo-actuated ICSI. Haploid androgenetic embryos were cultured to the 8-cell stage. Androgenetic blastomeres with GFP expression were coated with inactivated Sendai-virus and grafted to an activated recipient oocyte from another cohort. Cleavage parameters of reconstructed zygotes were compared to controls. Blastocysts were transferred into the uterine cavity of a 2.5-day post-coitus CD-1 surrogate. Main results and the role of chance A total of 421 oocytes were enucleated with a survival rate of 98.6%. The ooplasts underwent ICSI; 351 (84.6%) constructs developed a single male pronucleus 4-6h post-ICSI, and 208 (59.3%) constructs cleaved to the 8-cell stage. Parthenogenic activation by calcium ionophore was successful in 96.4% of the recipient oocytes, confirmed by the appearance of a single female pronucleus. A total of 148 haploid androgenotes with GFP expression were grafted onto corresponding activated oocytes; 145 successfully fused. Up to 2 unused sibling haploid blastomeres per embryo were genotyped using whole exome sequencing by a reference laboratory. A total of 481 untreated oocytes were inseminated, yielding 437 control zygotes. The cleavage of reconstructed embryos into the 2-cell (96.4%), 4-cell (94.3%), and 8-cell stage (91.4%) was comparable to that of control conceptuses (97.5%, 93.8%, and 92.7% respectively). Although morula compaction (80.0%) and blastulation (60.7%) were lower than control (89.2% and 80.8%, P <0.001, respectively), morphokinetic parameters were comparable. A total of 45 blastocysts were transferred into 5 recipient mice; 8 blastocysts implanted (17.8%) in 2 surrogates, yielding 5 live offspring (11.1%) weighing 1.51±0.22g. All live offspring expressed GFP and were confirmed fertile once reaching adulthood. Limitations, reasons for caution Although copies of gamete substitutes were generated that could be genotyped and used to produce live offspring with the desired phenotype in a mouse model, the system needs to be optimized and proven reproducible in humans, specifically in regard to the role of the sperm centrosome. Wider implications of the findings Once our proposed technique is implemented in humans, it may benefit patients to control germline heterozygosity by allowing genotyping of identical copies of the gametes. This technique, when used in conjunction with heritable genomic editing, can obviate the inherently highly condensed sperm genome to provide genome-edited gamete substitutes. Trial registration number N/A

P-270 Assisted gamete treatment to pinpoint acquired meiotic maturity and overcome oocyte activation deficiency contributed by both gametes

Abstract Study question How can we treat couples with complete and persistent fertilization failure with ICSI linked to a combination of oocyte- and sperm-related oocyte activation deficiency (OAD)? Summary answer By targeting spindle presence, we optimized oocyte response to chemical activation and enhanced fertilization. Genomic assessment confirmed gamete contribution. What is known already Total fertilization failure occurs in 1-3% of all intracytoplasmic sperm injection (ICSI) cases. In sperm-factor OAD, the lack of phospholipase C zeta (PLCζ) prevents the spermatozoon from initiating downstream calcium oscillation in the oocyte. In these cases, assisted gamete treatment (AGT), which exposes gametes to calcium ionophore, has been adopted to artificially trigger the influx of calcium ions and has been shown to effectively improve fertilization. However, AGT is limited to triggering an intracytoplasmic calcium influx and still requires optimal ooplasmic maturity. Study design, size, duration Over the past 17 months, we identified couples with compromised PLCζ and reported persistent fertilization failure with ICSI despite AGT treatment. We then devised a treatment plan comprising an extended in vitro culture (IVC) to pinpoint meiotic oocyte maturity confirmed by the presence of a meiotic II spindle and followed by AGT post-ICSI. Genomic assessment was also carried out. Participants/materials, setting, methods Two couples with recurrent and total fertilization failure even after AGT were included. PLCζ expression was assessed using immunofluorescence on ≥ 200 cells/specimen with a 30% threshold. In the follow-up cycles, IVC was extended for at least 8 hours between retrieval and ICSI. Metaphase II spindles were visualized by Oosight®. AGT was performed by exposing both spermatozoa and oocytes to calcium ionophore. NGS was performed on spermatozoa to identify gene mutations involved in fertilization. Main results and the role of chance We identified 2 couples (couple A: 37-year-old female, 39-year-old male; couple B: 32-year-old female, 33-year-old male) with the following semen parameters: average volume of 2.6 ml, concentration of 82.0x106/ml, 44% motility, and normal morphology of 3%. The oocyte maturation rate was 76.3% (45/59) but resulted in zero fertilized out of a total of 45 MII oocytes injected. In-house PLCζ assessment revealed a deficiency of oocyte activation factor at 12.9%. AGT treatment alone failed to enhance fertilization on a subsequent cycle, resulting in 0% (0/8) and 5.6% (1/18) fertilization rates for couples A and B, respectively. Couple A then underwent 3 ICSI cycles with extended IVC and AGT; upon examination of nuclear maturity, 91.4% (32/35) of oocytes displayed normal metaphase II spindle and achieved an overall fertilization rate of 43.8% (14/32). To date, 12 blastocysts were cryopreserved. In couple B, 27 oocytes out of 34 retrieved presented normal metaphase II spindles after extended IVC; ICSI with AGT yielded a fertilization rate of 63.0% (17/27). All 17 zygotes were cryopreserved. Overall, our treatment improved fertilization to an overall rate of 52.5% (31/59, P <0.00001). Genomic assessment of spermatozoa identified gene mutations involved in fertilization (ADAM15, ADAM30) and calcium channel activity (CATSPER1). Limitations, reasons for caution Assisted gamete treatment can enhance fertilization in cases of deficiency in PLCζ. However, chemical activation requires a responsive ooplasm that has reached meiotic maturity. These rare cases require precise diagnoses and tailored treatment techniques to address each aspect of sperm- and/or oocyte-factor OAD. Wider implications of the findings Our study has demonstrated the usefulness of extended IVC by targeting spindle presence to enhance chemical responses to AGT. Our findings show that although calcium ionophore can trigger the release of intracellular calcium and allow fertilization, a fully mature ooplasm is required. Trial registration number N/A

P-238 Treatment options to overcome impaired fertilization and embryonic development caused by an oocyte-related deficiency in the Patl2-/- mouse model

Abstract Study question Can assisted oocyte activation (AOA) or spindle transfer (ST) overcome the lower fertilization and blastocyst rates observed in the Patl2-/- mouse model? Summary answer ST, but not AOA, has the potential to restore both normal fertilization and blastocyst rates in a mouse model with an oocyte-related deficiency. What is known already PATL2 is an RNA-transcriptional repressor involved in the regulation of maternal mRNAs during oocyte maturation. Mutations in PATL2 have been detected in patients with oocyte maturation arrest, reduced fertilization and embryo developmental arrest. Consequently, these patients are mostly referred to oocyte donation. Both AOA, inducing artificially calcium oscillations by calcium ionophores, and ST, replacing bad-quality oocyte cytoplasm with healthy cytoplasm, have been proposed to treat oocyte-related infertility. However, there is a lack of studies showing which oocyte-related infertility indications may benefit from which specific treatment. The Patl2-/- mouse model offers a unique opportunity to study the efficiency of these treatments. Study design, size, duration An experimental study using C57BL/6NTac-Patl2tm1a mice was conducted between April to December 2021. Breeding of heterozygous C57BL/6NTac-Patl2tm1a mice resulted in a total of 156 pups, from which 22 were homozygous C57BL/6NTac-Patl2tm1a ( Patl2-/-) females. Metaphase II (MII) oocytes were collected from Patl2-/- (test group) and Patl2+/+ (control group) females and used to evaluate different oocyte quality markers, as well as the efficiency of AOA and ST treatments to improve the oocyte-related subfertility observed in Patl2-/- mice. Participants/materials, setting, methods Four- to 12-week-old mice were subjected to ovarian hyperstimulation and oocyte collection, where oocyte maturation was assessed. Afterwards, MII oocytes were used to evaluate oocyte diameter, spindle abnormalities, activation rate (AR) and blastocyst rate (BR) after PIEZO-ICSI and calcium releasing capacity after SrCl2 exposure. Finally, Patl2-/- MII oocytes were treated with AOA, by direct exposure to SrCl2, or ST treatment, by transferring the Patl2-/- spindle to Patl2+/+ cytoplasm (previously enucleated) followed by SrCl2 exposure. Main results and the role of chance No difference in the number of cumulus-oocyte complexes (COCs) and mature MII oocytes was observed between 6 Patl2-/- mice (128 MII/159 COCs) and 5 Patl2+/+ mice (108 MII/132 COCs). However, Patl2-/- MII oocytes showed a significantly lower cytoplasm diameter (68,11 ± 2,25, n = 79) than Patl2+/+ oocytes (75,19 ± 2,09, n = 77, p-value<0,001), suggesting that Patl2 affects oocyte growth but does not seem to affect oocyte maturation. Nuclear maturity was not compromised either, as a similar percentage of oocytes with normal spindle was observed in Patl2-/- (48,4%, 15/31) and Patl2+/+ MII oocytes (50%, 18/36). Activation and blastocyst rates after PIEZO-ICSI were lower in Patl2-/- (AR = 45,5%, 5/11 and BR = 20,0%, 1/5) than in Patl2+/+(AR = 75,0%, 21/28 and BR = 52,4%, 11/21). In addition, total calcium released during oocyte activation after SrCl2 exposure was not different between Patl2-/- (AxF=6,88 AU, n = 30) and Patl2+/+ MII oocytes (AxF=5,04 AU, n = 28, p-value=0,087). In line with these results, AOA treatment did not improve activation and blastocyst rates in Patl2-/- oocytes (AR = 66,7%, 44/66 and BR = 45,5%, 20/44) compared to Patl2+/+ oocytes (AR = 96,5%, 110/114 and BR = 79,1%, 87/110, p-value<0,001). Nonetheless, when performing ST using Patl2-/- as spindle donors and Patl2+/+ as cytoplasm recipients, activation (8/8, 100,0%) and blastocyst rate (6/8, 75,0%) were restored to normal values. Limitations, reasons for caution This study describes preliminary results and is limited by its small sample size. In addition, we did not identify an increased number of immature oocytes and spindle abnormalities in Patl2-/- female mice in contrast to previously published reports. Wider implications of the findings ST could be offered to treat PATL2- related female-infertility caused by a cytoplasmic deficiency, to increase both fertilization and blastocyst rates. AOA would not be beneficial for these patients as calcium releasing capacity of Patl2-/- oocytes does not seem to be affected, suggesting a deficiency independent from calcium releasing machinery. Trial registration number Not applicable

P-060 Bubble-shaped Acrosome: Novel phenotype of Teratozoospermia Caused by Mutation in Actin-like 7A

Abstract Study question What’s the etiology of severe teratozoospermia characteristic as bubble-shaped acrosome (BSA)? Summary answer Severe teratozoospermia characterized as BSA caused by mutation (c.1024G>A) in actin-like 7A (ACTL7A). What is known already Teratozoospermia is a common cause of male infertility, defined by having a proportion of morphologically normal sperm at less than 4%. It exhibits aberrant sperm phenotypes in the head, neck, midpiece, and endpiece of sperm. Teratozoospermia with ephalic abnormalities are among the most severe and characteristic sperm defects. Some genetic factors are reported to be associated with ephalic abnormalities such as globozoospermia and macrozoospermia. However, other phenotypes and the causative genes of ephalic abnormalities, especially in acrosomal structure, and were largely unknown. Study design, size, duration Severe teratozoospermia were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya from Jan 2019 to Dec 2021. Participants/materials, setting, methods Whole-exome sequencing analysis was used to analyze the genetic factor of man. An Actl7a-mutated mouse model was generated by CRISPER-Cas9. Transmission electron microscopy was used to detect the abnormality of ultrastructure during acrosome biogenesis. Immunostaining was used to analyze the localization of ACTL7A and PLCζ. Immunoprecipitation followed by liquid chromatography-mass spectrometry (LC-MS) was used to select the differentially expressed proteins. ICSI with calcium ionophore exposure was performed in couple with ACTL7A mutation. Main results and the role of chance We found a man with severe teratozoospermia characterized as BSA carrying a mutation (c.1024G>A) in ACTL7A. Homozygous Actl7a-mutated male mice were sterile, and all of sperm showed acrosomal abnormalities. During acrosomal biogenesis, it detected the acrosome detach from the nuclear in Actl7a-mutated mice. Furthermore, mutant ACTL7A failed to attach to the acroplaxome and was discharged by cytoplasmic droplets, which led to the absence of ACTL7A in mature sperm. The mutant sperm failed to activate the oocyte, and PLCζ discharge accompanied by ACTL7A was observed, leading to total fertilization failure (TFF). Immunoprecipitation followed by LC-MS showed that several differentially expressed proteins participate in acrosome assembly and actin filament organization. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF in a couple with an ACTL7A mutation. Limitations, reasons for caution More cases are needed to demonstrate the relationship between mutation and phenotype. Wider implications of the findings Our study defined a novel phenotype of the acrosomal abnormality characterized as BSA and revealed the underlying mechanism of mutation in ACTL7A and provided a genetic marker and a therapeutic option for male infertility. Trial registration number Not Applicable

Calcium Ionophore A23187 treatment to rescue unfertilized oocytes: a prospective randomized analysis of sibling oocytes

Research questionCan 1-day old human unfertilized oocytes activate and blastulate after exposure to calcium ionophore (Ca.I) A23187? DesignProspective randomized trial analysis of sibling oocytes. Seventy unfertilized sibling oocytes from 24 couples were randomly split into two groups. In the treatment group, 35 oocytes were cultured with 5-µM Ca.I A23187 for 10 min, washed and cultured until day 6 of development (D+6). The remaining 35 oocytes (control group) were similarly cultured until D+6. Activation, cleavage and blastulation rates were compared between the two groups. ResultsComparable activation rates were observed in the oocytes incubated with Ca.I A23187 and in the control group (11.4% versus 17.1%; P = 0.49). The cleavage rate observed was 45.7% in both groups. None of the embryos reached blastocyst stage. ConclusionsActivation and cleavage can occur in unfertilized oocytes after the diagnosis of failure to fertilize. Unfortunately, the prevalence of activation is not affected by exposure to Ca.I A23187. Additionally, these embryos have no tangible reproductive potential as they arrest before reaching the blastocyst stage.

Calcium Ionophore Supplementation in In vitro Fertilization Medium towards Fertilization Rates of Kacang Goats (Capra hircus)

In vitro fertilization is assisted reproduction technology that can overcome infertility problems. This method can produce embryos with high quality in large quantities. In its application, the success rate of IVF is still relatively low. This is due to the poor quality of oocytes and spermatozoa, which can cause IVF failure. The absence of oocyte activation during fertilization also results in failure of fertilization. Calcium ionophore is known to be able to increase the oocyte activation process. This study aims to prove that Calcium Ionophore can increase the rate of fertilization in goat oocytes in vitro. This study was divided into two groups: the control group was fertilized oocytes using a medium without Calcium ionophore supplementation, the treatment group was fertilized oocytes using a medium supplemented with Calcium ionophore at a dose of 5.2 µl/ml. Fertilization rates are observed based on the formation of zygote or two pronuclear (2PN) in the cytoplasm of oocytes and observed under an inverted microscope. The data obtained were analyzed using the T test. The results of the study after being analyzed statistically showed that there were significant differences between the control group and the treatment group (P<0.05). Fertilization rates in the treatment group (93.12±13.7) showed higher results than the control group (65.96±28.36). Based on these results it can be concluded that calcium ionophore supplementation at a dose of 5.2 µl/ml in the fertilization medium can increase oocyte activation thereby increasing the rate of oocyte fertilization of the goat nuts.

Abstract 1350: The role of phosphatidylserine scramblases in tumor progression

Abstract Introduction: Phosphatidylserine (PS), an anionic phospholipid, is asymmetrically distributed in the inner leaflet of the phospholipid bilayer in normal cells under homeostasis. However, PS is constitutively externalized in the tumor microenvironment (TME); on viable cells that undergo stress, such as hypoxic tumor cells, stressed tumor vascular endothelial cells, as well as on cells undergoing apoptosis. This high PS harbors a pro-tumorigenic micro-environment, as myeloid cells expressing PS receptors (Mertk, Tyro3 and Axl) engage with externalized PS and secrete immunosuppressive cytokines. PS is externalized on viable cells by a calcium activated scramblase TMEM16F and on apoptotic cells by a caspase activated scramblase called Xkr8. Here, to study the mechanisms by which PS is externalized in the TME, we hypothesize that TMEM16F and Xkr8 are the major scramblases responsible for creating an immune suppressive milieu in the tumor microenvironment. Methods: To study the mechanisms of PS dysregulation in the tumor microenvironment, TMEM16F and Xkr8 were knocked out in E0771 murine breast cancer cell line using CRISPR/Cas9. The knockouts were validated by western blotting, q-RT-PCR and surveyor assays. Results: TMEM16F KO E0771 cells express lower basal levels of PS compared to WT E0771 cells when detected by Annexin V staining by flow cytometry. TMEM16F KO cells are also less responsive to activation by calcium ionophore. TMEM16F KO, Xkr8 KO and WT E0771 cells have similar viability and migration patterns in response to Gas6, indicating that the cells themselves do not have altered oncogenic potential. TMEM16F KO E0771 cells injected into the inguinal mammary gland fat pad of C57BL6 syngeneic mice showed modest but significantly reduced tumor growth compared to the E0771 WT tumors, suggesting that TMEM16F mediated PS externalization could play a role in exacerbating tumor progression. Conclusion: TMEM16F driven PS externalization contributes to tumor growth. The effect of TMEM16F KO on immune regulation of the tumor milieu will be discussed. These data corroborate previous studies targeting PS in tumors therapeutically using anti-PS antibodies and suggests a role for scramblases in pleiotropic immune-suppression in the tumor. Citation Format: Varsha Gadiyar, Viralkumar Davra, David Calianese, Kevin Lahey, Raymond B. Birge. The role of phosphatidylserine scramblases in tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1350.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n= 15) and normozoospermic (n= 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Inhibition of NETosis by a Nuclear-Penetrating Anti-DNA Autoantibody.

Nuclear-penetrating anti-DNA autoantibodies have therapeutic potential as delivery agents and in targeting DNA and the DNA damage response (DDR). Derivatives of such Abs have advanced to human testing in genetic disease and are in preparation for oncology clinical trials. DNA release associated with neutrophil extracellular traps (NETs) contributes to immunity, inflammation, and the pathophysiology of multiple diseases. The DDR contributes to mechanisms of NETosis, and we hypothesize that anti-DNA autoantibodies that localize into live cell nuclei and inhibit DNA repair will suppress release of NETs by activated neutrophils. In the current study we evaluated the impact of a nuclear-penetrating anti-DNA autoantibody that interferes with the DDR on decondensation and release of DNA and NETs by activated human granulocyte-like differentiated PLB-985 cells and neutrophils isolated from C57BL/6 mice. The response of cells pretreated with control or autoantibody to subsequent stimulators of NETosis, including PMA and the calcium ionophore ionomycin, was evaluated by DAPI and SYTOX Green stains, measurement of DNA release, analysis of histone citrullination by Western blot, or visualization of NETs by immunostaining and confocal fluorescence microscopy. Autoantibody treatment of the cells yielded significant inhibition of NADPH oxidase-dependent and independent NETosis. These findings establish the concept of nuclear-penetrating anti-DNA autoantibodies as modulators of neutrophil biology with potential for use in strategies to suppress NETosis.

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.