Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts.

SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation.

Attitude of Belgian women towards enucleated egg donation for treatment of mitochondrial diseases and infertility.

RESEARCH QUESTION: What is the attitude of Belgian women of reproductive age towards enucleated egg donation? Does the willingness of women to donate differ when they would donate enucleated or whole eggs? DESIGN: In 2022, an online survey was conducted among a representative sample of 1000 women in Belgium aged 18-50 years. The item on willingness to anonymously donate enucleated eggs was dichotomized into those willing to donate and those not willing to donate or uncertain. RESULTS: No statistically significant difference was found between the willingness to donate enucleated eggs and whole eggs (whether anonymously or identifiably). Anonymity, however, affected the willingness to donate, with considerably fewer women willing to donate identifiably. The respondents were divided about their parental status if they were to donate enucleated eggs, with less than one-half (44%) not considering themselves to be a genetic mother. Women willing to donate enucleated eggs anonymously were less likely to view themselves as a genetic mother of the child compared with others. Fewer than one in five considered the technique unacceptable because the resulting child would carry genetic material of three persons. CONCLUSIONS: Women in the general population did not show a greater willingness to donate enucleated eggs than whole eggs. The fact that the respondents were strongly divided on whether or not they would consider themselves to be a genetic mother of the resulting child may explain this result. Other factors, such as the potential high risk for the child, may also have contributed to less willingness.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targeted GENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.

Human germline nuclear transfer to overcome mitochondrial disease and failed fertilization after ICSI.

PURPOSE: Providing additional insights on the efficacy of human nuclear transfer (NT). Here, and earlier, NT has been applied to minimize transmission risk of mitochondrial DNA (mtDNA) diseases. NT has also been proposed for treating infertility, but it is still unclear which infertility indications would benefit. In this work, we therefore additionally assess the applicability of NT to overcome failed fertilization. METHODS: Patient 1 carries a homoplasmic mtDNA mutation (m.11778G > A). Seventeen metaphase II (MII) oocytes underwent pre-implantation genetic testing (PGT), while five MII oocytes were used for spindle transfer (ST), and one in vitro matured (IVM) metaphase I oocyte underwent early pronuclear transfer (ePNT). Patients 2-3 experienced multiple failed intracytoplasmic sperm injection (ICSI) and ICSI-assisted oocyte activation (AOA) cycles. For these patients, the obtained MII oocytes underwent an additional ICSI-AOA cycle, while the IVM oocytes were subjected to ST. RESULTS: For patient 1, PGT-M confirmed mutation loads close to 100%. All ST-reconstructed oocytes fertilized and cleaved, of which one progressed to the blastocyst stage. The reconstructed ePNT-zygote reached the morula stage. These samples showed an average mtDNA carry-over rate of 2.9% ± 0.8%, confirming the feasibility of NT to reduce mtDNA transmission. For patient 2-3 displaying fertilization failure, ST resulted in, respectively, 4/5 and 6/6 fertilized oocytes, providing evidence, for the first time, that NT can enable successful fertilization in this patient population. CONCLUSION: Our study showcases the repertoire of disorders for which NT can be beneficial, to overcome either mitochondrial disease transmission or failed fertilization after ICSI-AOA.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization.

BACKGROUND: The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification. METHODS: We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%. RESULTS: A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow. CONCLUSION: dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).

PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence.

Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca2+ oscillations that normally initiate embryogenesis. Consistent with this, recombinant PLCζ induces Ca2+ oscillations in eggs and debilitating mutations in the PLCZ1 gene are associated with infertility in men. However, there has been no evidence that knockout of the gene encoding PLCζ abolishes the ability of sperm to induce Ca2+ oscillations in eggs. Here, we show that sperm derived from Plcz1-/- male mice fail to trigger Ca2+ oscillations in eggs, cause polyspermy and thus demonstrate that PLCζ is the physiological trigger of these Ca2+ oscillations. Remarkably, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, Plcz1-/- males are subfertile but not sterile, suggesting that in the absence of PLCζ, spontaneous egg activation can eventually occur via an alternative route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring. PLCζ-null sperm now make it possible to resolve long-standing questions in fertilization biology, and to test the efficacy and safety of procedures used to treat human infertility.

Vitrification negatively affects the Ca2+-releasing and activation potential of mouse oocytes, but vitrified oocytes are potentially useful for diagnostic purposes.

RESEARCH QUESTION: To what extent does vitrification affect the Ca2+-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa? DESIGN: The effect of mouse oocyte vitrification on Ca2+ dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca2+ store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca2+ pattern induced by cryoprotectant exposure was determined. RESULTS: After human sperm injection into mouse oocytes, Ca2+ dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca2+ dynamics in response to SrCl2 or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl2 exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca2+ store content. Oocytes rapidly recovered during warming and recovery in Ca2+-containing media; a threshold area under the curve of Ca2+ dynamics to obtain activation rates above 90% was determined. CONCLUSIONS: Vitrified-warmed mouse oocytes display reduced Ca2+-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca2+-signalling machinery in vitrified-warmed human oocytes is required.

Human blastocyst outgrowths recapitulate primordial germ cell specification events.

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2- and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.

Assessment of the calcium releasing machinery in oocytes that failed to fertilize after conventional ICSI and assisted oocyte activation.

RESEARCH QUESTION: Can oocyte-related activation deficiencies be evaluated in oocytes that failed to fertilize after intracytoplasmic sperm injection (ICSI) combined with assisted oocyte activation (AOA)? DESIGN: Evaluation of the spindle-chromosome complexes and intracellular distribution of inositol trisphosphate type 1 receptors (IP3R1) in in-vitro matured (IVM) and failed-to-fertilize oocytes from patients undergoing AOA. Assessment of the oocyte-related Ca2+ releasing capacity in response to Ca2+ ionophores and sperm microinjection in oocytes that failed to fertilize after ICSI or ICSI-AOA. RESULTS: IVM oocytes from patients undergoing conventional ICSI (control) and ICSI-AOA (study group) revealed a similar normalcy of spindle-chromosome complexes and distribution patterns of IP3R1. Failed-to-fertilize oocytes from both groups showed significant differences in proportion of normal or abnormal spindle-chromosome complex conformations. However, migration of IP3R1 was identified in a higher proportion of failed-to-fertilize oocytes after ICSI-AOA than after conventional ICSI. It was further observed that oocytes which failed to fertilize, either after ICSI or ICSI-AOA, mostly retain their capacity to respond to stimuli such as exposure to Ca2+ ionophores or to sperm microinjection. CONCLUSIONS: Evaluation of spindle-chromosome normalcy and distribution of IP3R1 does not help identify the presence of Ca2+ releasing deficiencies in these oocytes. However, oocyte Ca2+ analysis adds value in identifying Ca2+ releasing incapacity of oocytes that failed to fertilize after ICSI or ICSI-AOA. Some patients experiencing fertilization failure after ICSI-AOA present with a suspected activation deficiency downstream of the Ca2+ machinery, which cannot be overcome by ICSI-AOA based on the use of Ca2+ ionophores.

Single Ca2+ transients vs oscillatory Ca2+ signaling for assisted oocyte activation: limitations and benefits.

Oocyte activation is a calcium (Ca2+)-dependent process that has been investigated in depth, in particular, regarding its impact on assisted reproduction technology (ART). Following a standard model of signal transduction, Ca2+ drives the meiotic progression upon fertilization in all species studied to date. However, Ca2+ changes during oocyte activation are species specific, and they can be classified in two modalities based on the pattern defined by the Ca2+ signature: a single Ca2+ transient (e.g. amphibians) or repetitive Ca2+ transients called Ca2+ oscillations (e.g. mammals). Interestingly, assisted oocyte activation (AOA) methods have highlighted the ability of mammalian oocytes to respond to single Ca2+ transients with normal embryonic development. In this regard, there is evidence supporting that cellular events during the process of oocyte activation are initiated by different number of Ca2+ oscillations. Moreover, it was proposed that oocyte activation and subsequent embryonic development are dependent on the total summation of the Ca2+ peaks, rather than to a specific frequency pattern of Ca2+ oscillations. The present review aims to demonstrate the complexity of mammalian oocyte activation by describing the series of Ca2+-linked physiological events involved in mediating the egg-to-embryo transition. Furthermore, mechanisms of AOA and the limitations and benefits associated with the application of different activation agents are discussed.

Patients with a high proportion of immature and meiotically resistant oocytes experience defective nuclear oocyte maturation patterns and impaired pregnancy outcomes.

Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.

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.

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells.

We present a fully defined culture system (adapted Essential8TM [E8TM ] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8TM medium was modified by adding (1) l-proline, (2) l-ornithine, (3) Nω -hydroxy-nor-l-arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l-ornithine, followed by 3.5 mM l-proline and by lowering the arginine concentration in the medium to 99.5 µM. No major changes in pluripotency and cell amount could be observed for the adapted E8TM media with ornithine and proline. However, our subsequent ion mobility assisted data-independent acquisition (high-definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion.

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism.

Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1-5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.

Exogenous supplementation of Activin A enhances germ cell differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) derived in the presence of Activin A (ActA) demonstrate an increased differentiation propensity toward the germ cell lineage. In addition, mouse epiblast stem cells and mouse epiblast-like cells are poised toward germ cell differentiation and are derived in the presence of ActA. We therefore investigated whether supplementation with ActA enhances in vitro hESC differentiation toward germ cell lineage. ActA up-regulated early primordial germ cell (PGC) genes STELLA/DPPA3 (developmental pluripotency associated 3) and tyrosine kinase receptor cKIT in both ActA-derived and standard-derived hESCs indicating its role in priming hESCs toward the PGC lineage. Indeed, ActA plus bone morphogenic protein 4 (BMP4) strongly increased germ cell differentiation potential of hESCs based on the high expression of late PGC markers DAZL (deleted in azoospermia-like) and VASA/DDX4 (DEAD-box polypeptide 4) at mRNA and protein level. Hence, the combination of ActA with BMP4 provides an additional boost for hESCs to develop into postmigratory germ cells. Together with increased VASA expression in the presence of ActA and BMP4, we also observed up-regulation of endoderm-specific genes GATA4 (GATA binding protein 4) and GATA6. Finally, we were able to further mature these in vitro-derived PGC-like cells (PGCLCs) by culturing them in in vitro maturation (IVM) medium, resulting in the formation of germ cell-like clusters and induction of meiotic gene expression. In conclusion, we demonstrate for the first time a synergism between ActA and BMP4 in facilitating germ cell-directed differentiation of hESCs, which is enhanced by extended culture in IVM medium, as shown by cytoplasmic VASA-expressing PGCLCs. We propose a novel relationship between the endoderm and germ cell lineage during hESC differentiation.

PLCζ disruption with complete fertilization failure in normozoospermia.

PURPOSE: Intracytoplasmic sperm injection (ICSI) is widely used to achieve fertilization in the presence of severe male factor, resulting in high fertilization rates. Nevertheless, 1-3 % of couples experience complete fertilization failure after ICSI. When a male factor is identified, assisted oocyte activation (AOA) can help overcome fertilization failures. The objective of this study is to describe a case of repeated complete fertilization failures after ICSI with donor oocytes, and to investigate the molecular and functional aspects of phospholipase C zeta (PLCζ) protein in the patient semen. METHODS: The patient was a normozoospermic male who had previously fathered, through natural conception, four children by a different partner. Molecular and functional analysis of sperm-specific PLCζ in the patient and control samples by means of gene sequencing, immunocytochemistry, Western blot, mouse oocyte activation test (MOAT), and mouse oocyte calcium analysis (MOCA) were used. RESULTS: PLCζ expression levels and distribution were significantly disrupted, although MOAT and MOCA did not indicate a decrease in activation ability. CONCLUSIONS: Normozoospermic males can have disrupted expression and distribution of PLCζ, and reduced activation ability after ICSI in human oocytes, despite their normal activation potential in functional testing using mouse oocytes. Discrepancy among molecular and functional data might exist, as mutations in the gene sequence may not be the only cause of alteration in PLCζ protein related to activation failures.