Activation Rates Of Oocytes Research Articles

P-740 Monogenic and digenic variants in male PLCZ1, ACTL7A and ACTL9 genes cause fertilization failure after ICSI

Abstract Study question What is the frequency of PLCZ1, ACTL7A and ACTL9 variants in male patients suffering from fertilization failure (FF) after ICSI? Summary answer Male patients with FF after ICSI exhibit variants in PLCZ1 (27,3%), ACTL7A (13,6%) and ACTL9 (4,5%). Some patients present PLCZ1 variants combined with ACTL7A/ACTL9 variants. What is known already FF after ICSI has been often attributed to phospholipase C zeta (PLCζ) deficiency, and consequently to insufficient calcium release, necessary for fertilization. PLCZ1 genetic variants have been detected in one-third of males with FF. Recently, actin-like 7A (ACTL7A) and 9 (ACTL9) variants were also identified, but larger cohort studies are lacking. ACTL7A/ACTL9 variants alter the acrosome structure, resulting in reduced and abnormally localized PLCζ. Assisted oocyte activation (AOA), by calcium ionophore, restores fertilization in patients with PLCZ1 variants, and could also benefit patients with ACTL7A/ACTL9 defects. This study aimed to expand the spectrum of known PLCZ1, ACTL7A and ACTL9 variants. Study design, size, duration A prospective study was conducted from June 2020 to December 2022 including 22 male patients (P1-P22) with FF after ICSI. Patients were only recruited when they had a mean fertilization rate ≤33,33% in at least 1 ICSI cycle. All patients donated a saliva sample for genetic screening of PLCZ1, ACTL7A and ACTL9 genes and a sperm sample for the mouse oocyte activating test (MOAT). All couples with FF after ICSI were offered an AOA treatment. Participants/materials, setting, methods Genetic screening was performed via next-generation sequencing. Identified variants were classified employing Varsome and confirmed by bidirectional Sanger sequencing. Patients in which uncertain significance (VUS), likely pathogenic or pathogenic variants were found underwent additional diagnostic tests, such as the study of the sperm calcium releasing pattern in mouse (MOCA) and in in vitro matured (IVM) human (HOCA) oocytes, immunostaining of PLCζ and ACTL7A, and the analysis of the sperm morphology by transmission electron microscopy (TEM). Main results and the role of chance Genetic screening revealed heterozygous PLCZ1 variants, a novel variant p.Ile379Thr in P10 and the previously published variant p.His233Leu in three patients (P9, P19 and P20). In addition, two novel homozygous ACTL7A variants were detected, p.Ser364GlnfsTer9 in P1 and p.Gly214Ser in P22. Interestingly, digenic variants were observed in P6 (PLCZ1 p.Ile74Thr and ACTL7A p.Tyr183His) and in P8 (PLCZ1 p.His233Leu and ACTL9 p.Arg271Pro). All patients with identified variants showed a high oocyte activation rate (>70%) after MOAT, except P1 and P22 which activation rate was <25%, indicating a more detrimental sperm-related deficiency. While MOCA only detected deficient calcium release in P1 (and not for P6, P8, P9 and P10), HOCA revealed absence of calcium oscillations in all patients analyzed (P1, P8, P9 and P10), except for P6. Immunostaining experiments in P1 revealed no ACTL7A signal and decreased PLCζ signal in patient sperm. Moreover, TEM analysis in P1 confirmed acrosome detachment from nuclear membrane. Overall, AOA increased fertilization rate (from 7,23% to 53,04%) and pregnancy rate (from 7,14% to 46,15%) in patients with identified variants. Specifically, patients with monogenic PLCZ1 (P9 and P19) and ACTL7A (P1) variants, as well as digenic variants (P6 and P8) achieved a live birth after AOA. Limitations, reasons for caution Some patients have not undergone all diagnostic tests yet, thus functional data on all the found variants identified is still ongoing. In addition, patient recruitment for this study is continuing. Genetic screening was carried on exonic and flanking intronic regions, which might have missed additional variants in other intronic regions. Wider implications of the findings The MOAT/MOCA tests cannot detect some subtle sperm-related activation deficiencies. HOCA represents a more sensitive analysis but requires human oocytes. Therefore, genetic screening of PLCZ1, ACTL7A and ACLTL9 could be a faster and more cost-efficient diagnostic test for FF after ICSI. Furthermore, AOA treatment is very efficient for these patients. Trial registration number NA

Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187.

Despite the success of ICSI in treating severe male factor infertile patients, total fertilization failure (FF) still occurs in around 1-3% of ICSI cycles. To overcome FF, the use of calcium ionophores has been proposed to induce oocyte activation and restore fertilization rates. However, assisted oocyte activation (AOA) protocols and ionophores vary between laboratories, and the morphokinetic development underlying AOA remains understudied. A prospective single-center cohort study involving 81 in vitro matured metaphase-II oocytes from 66 oocyte donation cycles artificially activated by A23187 (GM508 CultActive, Gynemed) (n=42) or ionomycin (n=39). Parthenogenesis was induced, and morphokinetic parameters (tPNa, tPNf, t2-t8, tSB, and tB) were compared between the 2 study groups and a control group comprising 39 2PN-zygotes from standard ICSI cycles. Ionomycin treatment resulted in higher activation rates compared to A23187 (38.5% vs 23.8%, p=0.15). Importantly, none of the A23187-activated parthenotes formed blastocysts. When evaluating the morphokinetic dynamics between the two ionophores, we found that tPNa and tPNf were significantly delayed in the group treated by A23187 (11.84 vs 5.31, p=0.002 and 50.15 vs 29.69, p=0.005, respectively). t2 was significantly delayed in A23187-activated parthenotes when compared to the double heterologous control embryo group. In contrast, the morphokinetic development of ionomycin-activated parthenotes was comparable to control embryos (p>0.05). Our results suggest that A23187 leads to lower oocyte activation rates and profoundly affects morphokinetic timings and preimplantation development in parthenotes. Despite our limited sample size and low parthenote competence, standardization and further optimization of AOA protocols may allow wider use and improved outcomes for FF cycles.

Rapamycin improves the quality and developmental competence of in vitro matured oocytes in aged mice and humans.

Women over age 35 suffer from the inadequate number and poor quality of oocytes during assisted reproductive treatment, and making full use of the oocytes by in vitro maturation (IVM) is crucial. Rapamycin could improve the developmental competences of the post-maturation oocytes during in vitro aging, yet its effects on the IVM process of oocytes from an aged population were not clear. In this study, the immature oocytes from aged mice or older women underwent IVM with or without 10 nM rapamycin, followed by parthenogenetic activation or insemination and embryo culture. The developmental competence and quality of IVM oocytes in both groups were compared. The results showed that in aged mice, the maturation rate, activation rate, and cleavage rate of IVM oocytes were significantly elevated in the rapamycin group. Additionally, oocytes cultured with rapamycin presented decreased ROS levels, reduced chromosome aberration, and attenuated levels of γ-H2AX. During IVM of oocytes from older women, the GVBD rate, 24 h maturation rate, and 48 h maturation rate were increased in the rapamycin group, compared with those in the control group, although without significant differences. After intracytoplasmic sperm injection (ICSI) and further culture of human oocytes, the high-quality embryo rate in the rapamycin group was significantly elevated. Overall, rapamycin improved IVM outcomes of oocytes from aged mice and older women. The specific mechanism of the positive effects of rapamycin on IVM outcomes might be reducing ROS levels, mitigating DNA damage, and promoting developmental potential.

P-211 Significant differences in efficiency between two commonly used ionophore solutions for assisted oocyte activation (AOA): a prospective comparison of ionomycin and A23187

Abstract Study question Are there differences in oocyte activation rate and developmental morphokinetic after assisted oocyte activation (AOA) with either ionomycin or A23187? Summary answer Ionomycin produces greater oocyte activation rate than A23187 (43.1% vs 15.9%), while the morphokinetic pattern of parthenotes’ development is similar between both Ca2+ ionophores. What is known already Fertilization failure (FF) after ICSI is often due to a male factor, such as the alteration of sperm-borne oocyte activating factors or globozoospermia. AOA can restore fertilization rate by stimulating transient spikes of Ca2+ in the cytoplasm of the oocyte, but its efficiency and effect on embryo development are not fully characterized. Here, the efficiency of two AOA preparation and protocols commonly used in clinical practice is compared using parthenogenetically activated human oocytes. The objective is to identify the most appropriate protocol for AOA and to compare the morphokinetic pattern of the generated parthenotes up to expanded blastocyst (tB). Study design, size, duration Prospective study involving 120 human oocytes from 66 women from March 2019 to November 2021. Oocytes were activated with two AOA protocols after mock ICSI: i) A23187 (ready-to-use solution, GM508 CultActive (Gynemed) n = 69), and ii) Ionomycin (homemade solution, 10 µmol/L, n = 51). Oocyte activation and development were analyzed in both groups; further, the morphokinetic patterns were compared; videos of embryos obtained with donor oocytes and sperm (n = 39) were used as comparator of normal developmental kinetics. Participants/materials, setting, methods Oocytes were injected with latex microspheres to simulate ICSI, followed by AOA. A23187 was used according to manufacturer specification. For ionomycin, three incubations of seven minutes each were performed. Resulting parthenotes (1PN) were incubated in a time-lapse system for 160h. Activation and developmental rates, tPNf (pronucleus fading), t2, t3, t4, t5, t8 (from 1st division to 8-cell), tsB (blastulation onset) and tB (blastocyst expansion) were compared using Student’s T-test and ANOVA. Statistical significance: p-value <0.05. Main results and the role of chance Ionomycin resulted in a significantly higher oocyte activation rate (22/51, 43.1%) than A23187 (11/69, 15.9%), p = 0.0009. In the ionomycin group, 81.8% (18/22) of parthenotes reached the 2-cell stage, 45.5% (10/22) reached the 5-cell stage, and 18.2% (4/22) reached the pseudo-blastocyst stage, as expected for these pseudo-embryos lacking the contribution of the sperm. In the A23187 group, 54.5% (6/11) reached the 2-cell stage, 27.3% (3/11) the 5-cell stage, and none formed pseudo-blastocysts. While the number of parthenotes progressing through development is much lower for A23187, the expected poor development of human parthenotes past activation and corresponding low numbers did not allow to reach statistical significance (p > 0.05). tPNf was significantly different among the 3 groups compared: 47.4±37.5h (n = 11, A23187), 27.7±23.8h (n = 21, ionomycin) and 23.3±4.6h (n = 39, control), p = 0.0019; with ionomycin presenting an average tPNf similar to the one obtained by ICSI. Among activated oocytes, the morphokinetic pattern in the ionomycin and A23187 groups was very similar to the one obtained in the control group (p > 0.05 at all timings). As an example, the t5 in the 3 groups was 58.5±12.6h (n = 3, A23187), 45.6±21.8h (n = 10, ionomycin), and 49.5±12.4h (n = 34, control), p = 0.29. Limitations, reasons for caution The low number of parthenotes progressing past 2-cells limit the possibility to extract solid conclusions regarding the morphokinetic patterns after AOA. The efficiency of activation using the two tested protocol is however confirmed. Wider implications of the findings The use of homemade ionomycin solutions is an effective option for the treatment of fertilization failures where assisted oocyte activation is indicated. Caution should be exerted when using GM508 Cultactive to investigate fertilization failures of oocyte origin. Trial registration number not applicable

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

Rapamycin improves the quality and developmental competence of mice oocytes by promoting DNA damage repair during in vitro maturation

BackgroundIncreasing evidence has shown that the mammalian target of rapamycin (mTOR) pathway plays a critical role in oocyte meiosis and embryonic development, however, previous studies reporting the effects of rapamycin on oocyte IVM showed different or even opposite results, and the specific mechanisms were not clear.MethodsThe immature oocytes from female mice underwent IVM with rapamycin at different concentrations to select an optimal dose. The maturation rate, activation rate, subsequent cleavage and blastocyst formation rates, spindle assembly, chromosome alignment, mitochondrial membrane potential (MMP), ROS levels, and DNA damage levels were evaluated and compared in oocytes matured with or without rapamycin. In addition, the expression levels of genes associated with mTORC1 pathway, spindle assembly, antioxidant function, and DNA damage repair (DDR) were also assessed and compared.ResultsRapamycin at 10 nM was selected as an optimal concentration based on the higher maturation and activation rate of IVM oocytes. Following subsequent culture, cleavage and blastocyst formation rates were elevated in activated embryos from the rapamycin group. Additionally, oocytes cultured with 10 nM rapamycin presented decreased ROS levels, reduced chromosome aberration, and attenuated levels of γ-H2AX. No significant effects on the percentages of abnormal spindle were observed. Correspondingly, the expressions of Nrf2, Atm, Atr, and Prkdc in IVM oocytes were markedly increased, following the inhibition of mTORC1 pathway by 10 nM rapamycin.ConclusionRapamycin at 10 nM could ameliorate the developmental competence and quality of IVM oocytes of mice, mainly by improving the chromosome alignments. The inhibition of mTORC1 pathway, which involved in activating DDR-associated genes may act as a potential mechanism for oocyte quality improvement by rapamycin.

Treatment with MG132 prevents spontaneous activation of rat oocyte in culture and promotes embryonic development after intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an effective reproductive technique for obtaining rat offspring using preserved sperm with low or no motility. However, rat oocytes undergo spontaneous activation immediately after retrieval from the oviduct and poorly develop after ICSI unless it is performed quickly. Here, we evaluated whether treatment with MG132, the proteasome inhibitor, suppresses the spontaneous activation of oocytes before and during ICSI. After retrieval from the oviducts, the rate of development into morula and blastocyst from the oocytes cultured in vitro for 1 h prior to ICSI significantly decreased compared with that from the control oocytes subject to ICSI without culture (7% versus 36%). However, a higher proportion of oocytes treated with MG132 for 0, 1, and 3 h before and during ICSI developed into morulae and blastocysts (70%, 60%, and 52%, respectively). Offspring were obtained from oocytes treated with MG132 for 0 and 1 h before and during ICSI (percentage: 31%). Altogether, MG132 could suppress the spontaneous activation of rat oocytes and increase embryonic development after ICSI.

Delaying Effects of Prolactin and Growth Hormone on Aging Processes in Bovine Oocytes Matured In Vitro.

Aging processes accelerate dramatically in oocytes that have reached the metaphase-II (M-II) stage. The present work aimed to study the patterns and intracellular pathways of actions of prolactin (PRL) and growth hormone (GH) on age-associated changes in bovine M-II oocytes aging in vitro. To this end, we analyzed spontaneous parthenogenetic activation (cytogenetic assay), apoptosis (TUNEL assay), and the developmental capacity (IVF/IVC) of in vitro-matured oocytes after prolonged culturing. Both PRL and GH reduced the activation rate of aging cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs), and their respective hormone receptors were revealed in the ova. The inhibitor of Src-family tyrosine kinases PP2 eliminated the effects of PRL and GH on meiotic arrest in DOs, whereas the MEK inhibitor U0126 only abolished the PRL effect. Furthermore, PRL was able to maintain the apoptosis resistance and developmental competence of aging CEOs. The protein kinase C inhibitor calphostin C suppressed both the actions of PRL. Thus, PRL and GH can directly support meiotic arrest in aging M-II oocytes by activating MAP kinases and/or Src-family kinases. The effect of PRL in maintaining the developmental capacity of aging oocytes is cumulus-dependent and related to the pro-survival action of the protein kinase C-mediated signal pathway.

Production of mouse offspring from inactivated spermatozoa using horse PLCζ mRNA

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52–53% or 48–51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

Effects of assisted oocyte activation with calcium- ionophore and strontium chloride on in vitro ICSI outcomes.

Objective(s): Failed fertilization after intra-cytoplasmic sperm injection (ICSI) is mainly attributed to failed oocyte activation and can be overcome by artificial oocyte activation (AOA). The present study aims to compare in vitro outcomes of ICSI following two different assisted oocyte activation chemical procedures (SrCl2 and Ionomycin) in sibling oocytes of ICSI candidates.Materials and Methods: From March 2015 until February 2016, 105 infertile men with 99–100% abnormal sperm morphology, irrespective of sperm motility, concentration, or origin (semen or testicular) were included in this study. Out of these, 66 couples accepted to be included in the study group (Ionomycin/ SrCl2) and 39 couples requested routine AOA procedure (Ionomycin) as external control group. Primary outcomes of this study (fertilization, embryo quality, and post-implantation development) were compared between these groups.Results:Significantly higher oocyte activation (67.90±3.6% vs. 51.16±3.6%, P=0.004) and fertilization (65.23±3.63% vs. 49.65±3.63%, P=0.008) rates were observed in sibling oocytes treated with Ionomycin in comparison to the SrCl2 sibling group. Percentage of top quality embryos was insignificantly higher in SrCl2 groups compared to the Ionomycin group (29.90±4.27 vs. 20.65±4.05%, P=0.26).Conclusion: Ionomycin may be superior to SrCl2 for inducing oocyte activation. However, SrCl2 may be a more efficient means to support the development of better quality embryos following ICSI.

Role of autophagy in modulating post-maturation aging of mouse oocytes

Mechanisms for post-maturation oocyte aging (PMOA) are not fully understood, and whether autophagy plays any role in PMOA is unknown. To explore the role of autophagy in PMOA, expression of autophagosomes and effects of the autophagy (macro-autophagy) activity on PMOA were observed in mouse oocytes. Oocyte activation rates and active caspase-3 levels increased continuously from 0 to 18 h of in vitro aging. While levels of microtubule-associated protein light chain 3 (LC3)-II increased up to 12 h and decreased thereafter, contents of p62 decreased from 0 to 12 h and then elevated to basal level by 18 h. However, the LC3-II/I ratio remained unchanged following aging in different media or for different times. During in vitro aging up to 12 h, upregulating autophagy with rapamycin or lithium chloride decreased activation susceptibility, cytoplasmic calcium, p62 contents, oxidative stress, caspase-3 activation and cytoplasmic fragmentation while increasing developmental competence, LC3-II contents, LC3-II/I ratio, mitochondrial membrane potential, spindle/chromosome integrity and normal cortical granule distribution. Downregulating autophagy with 3-methyladenine (3-MA) produced opposite effects on all these parameters except cytoplasmic fragmentation. After 12 h of aging culture, however, regulating autophagy with either rapamycin/lithium chloride or 3-MA had no impact on oocyte activation susceptibility. It is concluded that autophagy plays an important role in regulating PMOA. Thus, during the early stage of PMOA, autophagy increases as an adaptive response to prevent further apoptosis, but by the late stage of PMOA, the activation of more caspases blocks the autophagic process leading to severer apoptosis.

Effect of two assisted oocyte activation protocols used to overcome fertilization failure on the activation potential and calcium releasing pattern

To assess the effect of two assisted oocyte activation (AOA) protocols with the use of two calcium (Ca(2+)) ionophores, ionomycin and A23187 (calcimycin), on the intracellular Ca(2+) level in mouse and human oocytes and the fertilization rates. Comparison of two Ca(2+) ionophores, ionomycin and A23187, regarding their capacity to increase the intracellular Ca(2+) level and to support subsequent oocyte activation and development. University hospital research laboratory. Patients undergoing intracytoplasmic sperm injection (ICSI) treatment and B6D2F1 mice. Assisted oocyte activation and microinjection of mouse and human oocytes with sperm. Measurement of the fertilizing and Ca(2+)-releasing ability of human sperm. Ionomycin was more potent than A23187 in provoking Ca(2+) increases in both mouse and human oocytes with significantly higher amplitude and area under the receiver operating characteristic curve. The oocyte activation rate was significantly higher when mouse oocytes were activated with the use of the ionomycin- rather than the A23187-based AOA protocol. Furthermore, oocyte activation rate was higher when human in vitro matured oocytes were activated with the ionomycin-based AOA protocol, but the difference did not reach statistical significance. In both mouse and human oocytes, the AOA protocol that used ionomycin was more efficient than the one that used A23187. Bearing in mind that mammalian fertilization is successful when the total dose of Ca(2+) released reaches a minimal threshold, the use of ionomycin for human AOA might be justified instead of the use of A23187.

Zearalenone exposure affects epigenetic modifications of mouse eggs.

Zearalenone (ZEA) is a mycotoxin produced by various Fusarium fungi, which has been shown to cause several cases of mycotoxicosis in farm animals and humans. However, there is no evidence regarding the effect of ZEA on mouse egg developmental competence. In this study, we found that the activation rate of maturated oocytes was affected in mice by ZEA treatment, indicating that ZEA affects egg developmental competence. And we explored possible mechanisms of low mouse maturated oocyte developmental competence after ZEA treatment from an epigenetic modification perspective. The fluorescence intensity analysis showed that 5-methyl cytosine level increased after ZEA treatment, indicating that the general DNA methylation level increased in the treated eggs. Moreover, histone methylations were also altered: H3K4me2 as well as H3K9me3 and H4K20me1, me2, me3 levels decreased in eggs that were cultured in high-dose ZEA medium. Thus, our results indicated that ZEA decreased egg developmental competence by affecting the epigenetic modifications.

Generation of mouse parthenogenetic embryonic stem cells and preliminary study of the differentiation ability to motor neurons

In this study, we generated embryonic stem cells from parthenogenetic embryos (PESCs), and induced them to differentiate to motor neurons, which could be an alternative source of histocompatible cells for replacement of therapy and theoretical foundation for studying the relationship of genome imprint and neural differentiation. The parthenogenetic activation rate of B6D2F1 mouse oocytes was 93.26%. We established eight parthenogenetic embryonic stem cell lines and the establishment rate from parthenogenetic embryos was 23.53%. The pluripotency marker Oct4, the cell surface marker SSEA-1, and alkaline phosphatase exhibited in PESCs. Karyotype analysis showed normal 40 chromosomes when examined at passages 10 and 30, which was in accordance with their oocyte origins. Three germinal layers were differentiated in vivo and in vitro. Mouse PESCs, which were treated by tretinoin and sonic hedgehog with extracellular matrix, could generate motor neurons that expressed the specific markers such as HB9 and Olig2.

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca2+ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca2+ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.

In vitro maturation of cumulus–partially enclosed immature human oocytes by priming with gonadotropin

To investigate the effect(s) of exogenous gonadotropin on the cytoplasmic and nuclear maturation of cumulus-partially enclosed immature human oocytes invitro derived from ovarian stimulation cycles. Experimental human study. University-based laboratory. Women, aged 26-35 years, with infertility secondary to male factors, underwent ovarian stimulation and intracytoplasmic sperm injection cycles using a long protocol of pituitary down-regulation. Cumulus-partially enclosed immature human oocytes that were retrieved from the stimulated cycles were collected at the time of intracytoplasmic sperm injection. The cumulus-partially enclosed immature human oocytes were allocated into two groups: [1] oocytes at the germinal vesicle (GV) stage; and [2] oocytes at the metaphase I (MI) stage. Each group was cultured invitro with and without gonadotropin supplements. Some metaphase II (MII) oocytes derived from the two groups were parthenogenetically activated and exposed to subsequent embryonic development for 168 hours invitro. Other MII oocytes were tested for meiotic apparatus analysis, including spindle morphology and chromosomal alignment, by immunofluorescence staining and scanning confocal microscopy. Oocyte maturation and activation rates, percentages of embryonic development, and spindle normalization were analyzed by χ(2) analysis, whereas oocyte maturation time was analyzed by one-way analysis of variance. For GV oocytes the maturation and activation rates were significantly higher during invitro maturation with supplementation with FSH/LH (68% vs. 60% and 82% vs. 62%, respectively). However, maturation time(22.78 ± 0.87 vs. 23.70 ± 0.94 hours), embryonic development (cleavage: 84% vs. 83%; four-cell: 72% vs. 66%; eight-cell: 48% vs. 43%; blastocyst: 5% vs. 7%), and meiotic apparatus normalization rates (55% vs. 61.1%) were similar. For MI oocytes there were no significant differences in the maturation rates (85% vs.84%), maturation time (14.81 ± 0.65 vs.15.73 ± 0.58 hours), activation rates (77% vs. 80%), embryonic development (cleavage rates: 80% vs. 83%; four-cell: 68% vs. 72%; eight-cell: 56% vs. 51%; blastocyst: 7% vs. 6%), and meiotic apparatus normalization rates (52.4% vs. 54.5%). CONLUSION(S): Gonadotropin supplements to the maturation medium play an important role in cumulus-partially enclosed oocytes at the GV stage; however, MI stage-derived oocytes from stimulated cycles fail to acquire improved maturity after invitro maturation. Furthermore, gonadotropin at the current concentration did not increase spindle or chromosomal abnormalities in MII oocytes maturated from either GV- or MI-stage oocytes invitro.

Expression ofinterferon regulatory factor-1 in the mouse cumulus-oocyte complex is negatively related with oocyte maturation

ObjectiveWe found previously that interferon regulatory factor (Irf)-1 is a germinal vesicle (GV)-selective gene that highly expressed in GV as compared to metaphase II oocytes. To our knowledge, the function of Irf-1 in oocytes has yet to be examined. The present study was conducted to determine the relationship between retinoic acid (RA) and RA-mediated expression of Irf-1 and the mouse oocyte maturation.MethodsImmature cumulus-oocyte-complexes (COCs) were collected from 17-day-old female mice and cultured in vitro for 16 hours in the presence of varying concentrations of RA (0-10 µM). Rate of oocyte maturation and activation was measured. Gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and cytokine secretion in the medium was measured by Bio-Plex analysis. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.ResultsThe rates of oocyte maturation to metaphase II and oocyte activation increased significantly with RA treatment (10 nM-1 µM). With 100 nM RA treatment, lowest level of Irf-1 mRNA and cumulus cell's apoptosis was found. Among 23 cytokines measured by Bio-Plex system, the substantial changes in secretion of tumor necrosis factor-α, macrophage inflammatory protein-1β, eotaxin and interleukin-12 (p40) from COCs in response to RA were detected.ConclusionWe concluded that the maturation of oocytes and Irf-1 expression are negatively correlated, and RA enhances the developmental competence of mouse immature oocytes in vitro by suppressing apoptosis of cumulus cells. Using a mouse model, results of the present study provide insights into improved culture conditions for in vitro oocyte maturation and relevant cytokine production and secretion in assisted reproductive technology.

244 INVOLVEMENT OF Na+/H+ ANTIPORTER 1 IN EMBRYONIC DEVELOPMENT OF RAT OOCYTES ACTIVATED BY ETHANOL

In mammalian oocytes, artificial activation is essential for further embryonic development of somatic cell nuclear transferred or round spermatid-injected oocytes. Unlike the protocol for mice, the optimal activation protocol for rat oocytes is not well established. Recently, we showed for the first time that a combined treatment with ethanol and 6-dimethylaminopurine (6-DMAP) efficiently induced activation of rat oocytes, resulted in a higher proportion of blastocyst formation as compared with other combined treatments or with the 6-DMAP treatment alone (Sano et al. 2009 Zygote 17, 29–36). Although the ethanol treatment has been used for oocyte activation not only in rats, but also in other species to date, it is unclear why the ethanol treatment is effective, especially in activating rat oocytes. Because it has been reported that alcohol alters intracellular pH via exchangers, transporters, or both on the membrane surface in the myocardium, it leads us to the possibility that the ethanol treatment regulates such exchangers, transporters, or both in rat oocytes. The aim of the present study was to clarify the role of Na+/H+ exchanges (NHE) in activating rat oocytes treated with ethanol. First, we examined which types of NHE are expressed in rat ovaries, oocytes, and parthenogenetic embryos. Expression of the NHE1 protein was also confirmed in both oocytes and parthenogenetic embryos, but not in NHE2 and NHE3. Treatment with 5-(N-ethyl-N-isopropyl)amiloride hydrochloride, an NHE inhibitor, did not affect the development of ethanol-activated oocytes to the 2-cell stage; however, it decreased the rate of blastocyst formation in a dose-dependent manner. The 5-(N-ethyl-N-isopropyl)amiloride hydrochloride treatment did not decrease development to either the 2-cell or the blastocyst stage of oocytes activated by 6-DMAP alone. Taken together, we conclude that ethanol treatment was involved in NHE1 activation, resulting in high developmental ability of rat oocytes. This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS (KAKENHI; 21780253) to J. I. The work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities, to J. I. and N. K.

P43. Several factors affect the developmental potential of rat somatic cell nuclear-transferred oocytes in vitro

Background: Since the first successful report of cloned mammals after nuclear transfer of somatic cells in sheep in 1997, cloned animals of various species have been produced. Generally, somatic cell nuclear transfer (SCNT) is performed with non-activated metaphase II (MII) oocytes as recipients, because activation of oocytes has adverse effects on the developmental potential of SCNT oocytes. Rat oocytes are spontaneously activated soon after ovulation, but Zhou et al. successfully cloned the first rat by SCNT using the proteasome inhibitor MG132 to inhibit spontaneous oocyte activation (2003. Science 302, 1179). To date, however, this is the only report of a cloned rat by SCNT, indicating that successful cloning of rat SCNT depends on other unknown factors as well. Here we examined several factors affecting the developmental potential of rat SCNT embryos aimed at establishing reliable procedures for successful rat SCNT. Methods and results: First, we examined the effect of MG132 on spontaneous activation of rat oocytes. The oocytes (from 3- to 5-week-old Sprague Dawley rats) maintained developmental potential after parthenogenetic activation for at least 4 h in 5 µM MG132-supplemented medium. Long exposure of SCNT oocytes to MG132 before activation affected the developmental potential due to spindle abnormalities. SCNT oocytes receiving fresh cumulus cells as nuclear donors with short-duration exposure to MG132 had a high cleavage rate, and several oocytes developed to the blastocyst stage, but the rate was very low, with or without treatment with the histone deacetylase inhibitor Trichostatin A (TSA), which improves the developmental potential of SCNT oocytes in mouse and bovine. When donor cells were cultured before SCNT, reconstituted oocytes developed to the 4-cell stage at a higher rate than non-cultured oocytes, even those of the same cell origin, and some oocytes developed to the blastocyst stage. To improve the developmental potential of rat SCNT oocytes, not only should spontaneous activation be avoided, but donor preparation, epigenetic modification, and in vitro culture conditions should also be examined.

Are zona pellucida laser drilling and polar body biopsy safe for in vitro matured oocytes?

Preconception diagnosis requires first polar body biopsy. When the hole in the zona pellucida is made with a laser beam, heat propagation could, like the biopsy itself, be deleterious. Our aim was to evaluate the effect of this technique on human in vitro matured oocyte and embryo development. One hunded fifty five retrieved immature oocytes from 75 women, matured in vitro, were distributed in 3 groups: 50 oocytes in a control group, without laser drilling and first polar body biopsy, 52 oocytes in a group with only laser drilling, and 53 oocytes in a group with both laser drilling and first polar body biopsy. Safety was evaluated using four criteria: [1] oocyte lysis rate, [2] oocyte activation rate, [3] oocyte development after calcium ionophore treatment, [4] and embryo chromosome breakage incidence after Tarkowski preparation. No difference in the four criteria was observed between the 3 oocyte groups. We did not find evidence of deleterious effect of laser drilling and first polar body biopsy on in vitro matured oocytes, according to our criteria.