Metaphase II Stage Research Articles

Canine oocyte nuclear maturation with Nano-ozone (NZS) supplementation: The alterations of antioxidant, and oxidant status and CDK1, cyclin B1 expressions

This study aims to evaluate the effects of nano-ozone solution (NZS) on canine oocyte nuclear maturation, associated with the alterations of antioxidant and oxidant status and cyclin-dependent kinase 1 (CDK1), cyclin B1 gene expressions. Oocytes were cultured in four distinct concentrations of NZS (0.5, 1, 2, and 5 µg/mL) and parthenogenetically activated. The rates of oocytes arrested at the Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (MI), and Metaphase II (MII) stages were statistically different among groups (P < 0.05). The oocytes cultured in 1 µg/mL NZS yielded the best oocyte maturation rate at the MI and MII stages; however, the lowest maturation and high degeneration rates were observed in Group E. The measurements of Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Ferric Reducing/Antioxidant Power assay (FRAP) were performed from IVM culture media. No statistical difference was observed in SOD and MDA results (P > 0.05). GSH levels were statistically significant between Group A-Group E (p = 0.003), Group B-Group E (p = 0.045), and Group E-Group D (p = 0.021). The culture media in Group D and Group E had high FRAP concentrations and significantly differed between groups (P < 0.05). CDK1, and cyclin B1 genes, which are subunits of maturation-promoting factor (MPF), are upregulated in Group B and Group C, while are downregulated in oocytes of Group E.This study showed that low, controlled doses of NZS (1 µg/mL) supplementation could improve the meiotic competence of canine oocytes and lead to positive response in expressions of CDK1 and cyclin B1 on the gene level.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclinB1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosisI. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphaseI (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclinB1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphaseII (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

Exogenous expression of PGC-1α during in vitro maturation impairs the developmental competence of porcine oocytes

Objectives of the current study were to examine the effects of exogenous expression of PGC-1α, which is a transcription factor responsive for controlling mitochondrial DNA (mtDNA) replication, mitochondria quantity control, mitochondrial biogenesis, and reactive oxygen species (ROS) maintenance, in porcine oocytes during in-vitro maturation (IVM) on the developmental competence, as well as mitochondrial quantity and function. Exogenous over-expression of PGC-1α by injection of the mRNA construct into oocytes 20 h after the start of IVM culture significantly increased the copy number of mtDNA in the oocytes, but reduced the incidences of oocytes matured to the metaphase-II stage after the IVM culture for totally 44 h and completely suppressed the early development in vitro to the blastocyst stage following parthenogenetic activation. The exogenous expression of PGC-1α also significantly induced spindle defects and chromosome misalignments. Furthermore, markedly higher ROS levels were observed in the PGC-1α-overexpressed mature oocytes, whereas mRNA level of SOD1, encoded for a ROS scavenging enzyme, was decreased. These results conclude that forced expression of PGC-1α successfully increase mtDNA copy number but led to increased ROS production, evidently by downregulation of SOD1 gene expression, inducement of spindle aberration/chromosomal misalignment, and consequently reduction in the meiotic and developmental competences of porcine oocytes.

In Vivo-Matured Oocyte Resists Post-Ovulatory Aging through the Hub Genes DDX18 and DNAJC7 in Pigs.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that "organelles", especially "mitochondria", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the "electron transport chain" and "cell redox homeostasis" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in "RNA binding", "mRNA metabolic process", etc., as well as in GO terms, and "spliceosome" and "nucleotide excision repair" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

PAK1-Dependent Regulation of Microtubule Organization and Spindle Migration Is Essential for the Metaphase I-Metaphase II Transition in Porcine Oocytes.

P21-activated kinase 1 (PAK1) is a critical downstream target that mediates the effect of small Rho GTPase on the regulation of cytoskeletal kinetics, cell proliferation, and cell migration. PAK1 has been identified as a crucial regulator of spindle assembly during the first meiotic division; however, its roles during the metaphase I (MI) to metaphase II (MII) transition in oocytes remain unclear. In the present study, the potential function of PAK1 in regulating microtubule organization and spindle positioning during the MI-MII transition was addressed in porcine oocytes. The results showed that activated PAK1 was co-localized with α-tubulin, and its expression was increased from the MI to MII stage (p < 0.001). However, inhibiting PAK1 activity with an inhibitor targeting PAK1 activation-3 (IPA-3) at the MI stage decreased the first polar body (PB1) extrusion rate (p < 0.05), with most oocytes arrested at the anaphase-telophase (ATI) stage. IPA-3-treated oocytes displayed a decrease in actin distribution in the plasma membrane (p < 0.001) and an increase in the rate of defects in MII spindle reassembly with abnormal spindle positioning (p < 0.001). Nevertheless, these adverse effects of IPA-3 on oocytes were reversed when the disulfide bond between PAK1 and IPA-3 was reduced by dithiothreitol (DTT). Co-immunoprecipitation revealed that PAK1 could recruit activated Aurora A and transform acidic coiled-coil 3 (TACC3) to regulate spindle assembly and interact with LIM kinase 1 (LIMK1) to facilitate actin filament-mediated spindle migration. Together, PAK1 is essential for microtubule organization and spindle migration during the MI-MII transition in porcine oocytes, which is associated with the activity of p-Aurora A, p-TACC3 and p-LIMK1.

Identification and molecular characterization of genes modulating progression of an oocyte from M-I to M-II in rat ovary.

To achieve oocyte competence for successful fertilization, bidirectional communication between oocyte and granulosa cells is crucial. The acquisition of meiotic competency in oocyte is facilitated by various regulatory genes however, expression pattern of these genes is not well documented during meiotic transition from Metaphase-I to Metaphase-II stage. Therefore, the present research analyzed the expression pattern of regulatory genes that are involved in the transition from M-I to M-II stages in rat oocyte. The analysis of the data was conducted by applying an array of bioinformatic tools. The investigation of gene group interactions was carried out by employing the STRING database, which relies on co-expression information. The gene ontology (GO) analysis was performed utilizing the comparative GO database. Functional annotation forGO and pathway enrichment analysis were performed for genes involved in networking.The GOobtained through computational simulations was subsequently validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The findings of our study suggest that there is a distinct gene expression pattern in both the oocyte and granulosa cells. This pattern indicates that oocyte-secreted factors, such as BMP15 and GDF9, play a crucial role in regulating the progression of the meiotic cell cycle from the M-I to M-II stages. We have also examined the level of mRNA expression of genes including CYP11A1, CYP19A1, and STAR, which are crucial for the steroidogenesis. It is fascinating to observe that the oscillatory pattern of specific key genes may hold significance in the process of in vitro oocyte maturation, specifically during the transition from the M-I to M-II stage. It might be useful for determining biomarker genes and potential pathways that play a role in attaining oocyte competency, thereby aiding in the assessment of oocyte quality for the purpose of achieving successful fertilization.

The association of RBX1 and BAMBI gene expression with oocyte maturation in PCOS women

BackgroundPolycystic ovarian syndrome (PCOS) is a common endocrine disorder that affects 6–20% of women of reproductive age. One of the symptoms of PCOS is hyperandrogenism, which can impair follicular development. This disruption can cause issues with the development of oocytes and the growth of embryos. Although the exact cause of PCOS is not yet fully understood, studying the gene expression pattern of cumulus cells, which play a crucial role in the maturation and quality of oocytes, could help identify the genes associated with oocyte maturation in PCOS women. Through indirect activation of APC/Cdc20, RBX1 enables oocytes to bypass the GV (germinal vesicles) stage and advance to the MII (metaphase II) stage. our other gene is the BAMBI gene which stimulates WNT signaling, that is a crucial pathway for healthy ovarian function. This study aims to explore the expression level of the RBX1 and BAMBI genes between GV and MII oocytes of PCOS and non-PCOS groups.MethodsIn this experiment, we gathered the cumulus cells of MII (38 cases and 33 control) and GV (38 cases and 33 control) oocytes from women with/without PCOS. Besides, quantitative RT-PCR was used to assess the semi-quantitative expression of BAMBI and RBX1.ResultsAccording to our research, the expression level of RBX1 and BAMBI in MII and GV cumulus cells of PCOS patients was significantly lower than that in non-PCOS ones.ConclusionThis research raises the possibility of RBX1 and BAMBI involvement in oocyte quality in PCOS women.

Long pentraxin 3 and vitamin D receptor mRNA expression pattern of cumulus granulosa cells isolated from PCOS oocytes at different stages of nuclear maturation

BackgroundA fine-tuned pro-inflammatory and anti-inflammatory balance in the follicular unit is essential for cumulus expansion and successful ovulation. While the long pentraxin 3 (PTX3) gene is required for the expansion of cumulus cells (CCs), ovulation, resumption of meiosis and fertilization, the vitamin D receptor gene (VDR-X2) is required for intra-follicle redox balance. This study was planned to determine the expression pattern of VDR-X2 and PTX3 mRNA in CCs isolated from germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes of PCOS patients with ovulatory dysfunction.MethodsThe relative expression of CC-PTX3 and CC-VDR-X2 mRNA were evaluated using qRT-PCR in a total of 79 CC samples collected from individual cumulus-oocyte complex of 40 infertile patients (20 PCOS and 20 non-PCOS normal responders) who underwent ovarian stimulation with the GnRH antagonist protocol.ResultsRelative PTX3 mRNA expressions of CCMI-control and CCMII-control showed 3- and 9-fold significant upregulation compared to CCGV-control, respectively. The relative PTX3 mRNA expression of CCMII-control increased approximately three fold compared to CCMI-control. Compared to CCGV-pcos, a 3-fold increase was noted in the relative PTX3 mRNA expression of CCMI-pcos and an approximately 4-fold increase in the PTX3 mRNA expression of CCMII-pcos. Relative PTX3 mRNA expression values of CCMII-pcos and CCMI-pcos were similar. A 6-fold upregulation of relative PTX3 mRNA and a 4-fold upregulation of VDR-X2 mRNA were detected in CCMII-control compared to CCMII-pcos. CC-VDR-X2 expression patterns of the PCOS and control groups overlapped with the CC-PTX3 pattern. Fertilization rates of the PCOS group exhibiting failed transcript expression were similar to normal responders.ConclusionThe fact that relative CC-PTX3 and CC-VDR mRNA expression does not increase during the transition from MI to MII stage in PCOS as in normal responders suggests that PTX3 and VDR expression may be defective in cumulus cells of PCOS patients with ovulatory dysfunction.

Vitrification of immature oocytes in pigs.

Cryopreservation of oocytes is an important technology for the in vitro gene banking of female germplasm. Although slow freezing is not feasible, porcine oocytes survive vitrification at high rates. Cryopreservation at the germinal vesicle stage appears to be more advantageous than that at the metaphase-II stage. Several factors are considered to affect the success of vitrification and subsequent utilization of immature porcine oocytes such as the device, the protocols for cryoprotectant application, warming, and the post-warming culture. Although live piglets could be obtained from vitrified immature oocytes, their competence to develop to the blastocyst stage is still reduced compared to their non-vitrified counterparts, indicating that there is room for further improvement. Vitrified oocytes suffer various types of damage and alteration which may reduce their developmental ability. Some of these can recover to some extent during subsequent culture, such as the damage of the cytoskeleton and mitochondria. Others such as premature nuclear progression, DNA damage and epigenetic alterations will require further research to be clarified and addressed. To date, the practical application of oocyte vitrification in pigs has been confined to the gene banking of a few native breeds.

Kisspeptin stimulates sheep ovarian follicular development in vitro through homologous receptors.

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene (KISS1R) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0-100 µg/ml) in the culture medium of PFs for 6 days. The cumulus-oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro. The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

Specc1l deficiency leads to abnormal oocyte meiosis and reduced blastocyst development in mouse.

This study aimed to investigate the potential role of the mouse Specc1l (Sperm antigen with calponin homology and coiled-coil domains 1 like), a microtubule and actin cytoskeleton-associated protein during oocyte meiotic maturation and its potential effects on preimplantation development. This study shows that the transcriptional levels of Specc1l did not significantly change from the germinal vesicle (GV) stage to the metaphase II (MII) stage, but maternal transcripts rapidly and gradually degraded after fertilization. Specc1l was detected in both the cytoplasm and GV, but not in the nucleolus-like body in the GV intact oocyte. At the MII stages, Specc1l was widely distributed in the cytoplasm but did not co-localize with chromatin. Knockdown of Specc1l expression in oocytes resulted in abnormal spindle morphology and misaligned chromosomes, as well as a decrease in the rate of polar body extrusion and a reduced developmental competence of oocytes, leading to decreased blastocyst formation rate. In conclusion, this study provides evidence that Specc1l plays a critical role in mouse oocyte meiotic maturation and early embryo development, specifically in proper bipolar spindle assembly and extrusion of the first polar body.

Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence.

Acquisition of germinal vesicle (GV) stage oocytes for fertility preservation (FP) offers several benefits over in vivo matured oocyte cryopreservation following ovarian stimulation, particularly for cancer patients necessitating immediate treatment. Two FP approaches for GV oocytes are available: vitrification before in vitro maturation (IVM) at the GV stage (GV-VI) or post-IVM at the metaphase II (MII) stage (MII-VI). The optimal method remains to be determined. In this study, mouse oocytes were collected without hormonal stimulation and vitrified either at the GV stage or the MII stage following IVM; non-vitrified in vitro matured MII oocytes served as the control (CON). The oocyte quality and developmental competence were assessed to obtain a better method for immediate FP. No significant differences in IVM and survival rates were observed among the three groups. Nevertheless, GV-VI oocytes exhibited inferior quality, including abnormal spindle arrangement, mitochondrial dysfunction, and early apoptosis, compared to MII-VI and CON oocytes. Oocyte vitrification at the GV stage impacted maternal mRNA degradation during IVM. In addition, the GV-VI group demonstrated significantly lower embryonic developmental competence relative to the MII-VI group. RNA sequencing of 2-cell stage embryos revealed abnormal minor zygotic genome activation in the GV-VI group. Vitrification at the GV stage compromised oocyte quality and reduced developmental competence. Consequently, compared to the GV stage, oocyte vitrification at the MII stage after IVM is more suitable for patients who require immediate FP.

O-182 The clinical value of rescued MI-oocytes. Retrospective analysis of 625 MI-oocytes versus 2124 sibling MII-oocytes obtained in 285 fresh donor ICSI cycles

Abstract Study question What are the developmental competence and clinical value in ICSI cycles of retrieved metaphase-I oocytes matured in vitro (rescued-MI) to the metaphase-II (MII) stage? Summary answer Rescued MI-oocytes showed lower developmental but similar post-implantation competence than their sibling MII-oocytes, thereby contributing to a + 9.5% relative-increase in the cumulative-live-birth rate per completed-cycle. What is known already MI-oocytes are typically excluded from ICSI. Nevertheless, some authors reported their use in cancer or poor responder patients. On average, 15-20% of the oocytes retrieved in IVF are immature, but 45% of them may mature within a few hours of culture. These rescued-oocytes showed lower developmental, chromosomal and reproductive competence. Regardless, they might be valuable for some patients. To date, most of the studies were conducted including patients using own oocytes, thus implicitly involving the bias of infertility and poor prognosis. Here we aimed at outlining the clinical value of rescued-MI oocytes in the context of egg donation cycles. Study design, size, duration Observational study conducted at a private IVF center including 284 fresh oocyte donation cycles (Jan-2020 to Dec-2021). Two hours after oocyte-retrieval, all MI-oocytes identified after denudation were cultured for 1-2 additional hour(s). If reaching the MII-stage, they were inseminated via ICSI with ejaculated sperm, as for sibling MII-oocytes. Single culture in continuous media was conducted. MI- and MII-derived embryonic cohorts were monitored and compared. One-hundred-fifty-three cycles in the same period had no MI-oocyte available. Participants/materials, setting, methods Two-hundred-eighty-four cycles were conducted from 272 recipients (42.1±4.0 years) with fresh oocytes retrieved from 215 donors (25.5±5.0 years). All sperm samples were ejaculated (42.3% fresh; 15% oligoasthenoteratozoospermic). The number of MI- and MII-oocytes after denudation was 2.2±1.3 (range:1-7) and 7.5±1.6 per cycle, respectively. 1.7±1.2 (0-6) MI-oocytes matured in vitro (rescue-rate: 78.1±33.4%) and were inseminated. Embryological data were compared between sibling MI- and MII-derived cohorts. Clinical data and relative contributions of MI-oocytes were also reported. Main results and the role of chance The mean fertilization rate per cohort of inseminated MI- and MII-oocytes were 54.3±43.0% and 74.4±18.1%, respectively (paired t-test&amp;lt;0.01). The mean blastulation rate per cohort of MI- and MII-derived zygotes were 53.1±44.4% and 59.1±25.9%, respectively (paired t-test=0.1). Overall, 0.5±0.7 (0-4) MI-derived blastocysts were obtained adding up to the 3.2±1.7 MII-derived ones, thereby resulting in a + 20.8±40.1% (0 to + 300%) relative increase per cycle. Overall, 268 (94%), 247 (89.7%), and 188 (66.2%) cycles had ≥1, ≥2 and ≥3 MII-derived blastocyst(s), respectively. These rates showed a + 1.1% (N = +3), +4.9% (N = +12) and +7.4% (N = +24) relative increase due to MI-derived blastocysts use. To date, 19 out of 63 MI-derived blastocysts transferred resulted in a live-birth (30.2%, 95%CI 19.6-43.2) versus 124 out of 399 MII-derived ones (31.1%, 95%CI 26.6-35.9; p = 0.99). We reported 4 miscarriages out of 27 clinical pregnancies (14.8%, 95%CI 4.8-34.6) versus 38 out 162 (23.5%, 95%CI 17.3-30.9, p = 0.45), respectively. To date, 70% (N = 199/284) of the cycles was concluded, with a cumulative-live-birth-rate of 61.3% (N = 122/199). This rate showed a + 9.5% (N = +18) relative increase due to MI-derived blastocysts use. Among 85 non-completed cycles, 19 (22.4%) have MI-derived blastocysts still available. Limitations, reasons for caution Retrospective study. A cost-effectiveness analysis is warranted, especially because MI-oocytes clinical use involves additional workload and costs. 31% (N = 44/140) of patients with ≥1 live-birth have supernumerary cryopreserved MI-derived blastocysts. Many cycles are not concluded. Gestational/perinatal outcomes were not reported. More data with own eggs and/or including chromosomal testing are needed. Wider implications of the findings Rescuing MI-oocytes might increase the number of blastocysts available per cycle, and possibly the cumulative-live-birth-rate. Nevertheless, this practice involves a higher workload and the production of more supernumerary blastocysts. Mostly poor prognosis couples might benefit from their use; therefore a couple-specific decision-making process is desirable. Trial registration number n/a

Effect of 2 Day vs. 3 Day Superstimulation Protocol on In Vivo Maturation of OPU derived oocytes in Buffaloes

The study was planned to evaluate effect of two different superstimulation protocols (2 Day vs. 3 Day) on in vivo maturation of oocytes in buffaloes. Buffaloes (n=8) were superstimulated twice using 2x2 design at an interval of 30-35 days using 200 mg and 300 mg FSH divided into four and six tapering doses (each dose of 50 mg) given over 2 and 3 days at 12 hourly intervals. Prostaglandin F2α analogue (500 μg Cloprostenol; i/m) at 72 and 84 hrs after start of superstimulatory treatment. Oocytes were retrieved from &gt; 6 mm follicles at 48 h after the last FSH injection by ovum pick up. Oocyte maturation was assessed by the degree of cumulus cell expansion and by Orcein staining of denuded oocytes. OPU of 60 and 91 follicles resulted in recovery of 38 and 50 cumulus oocyte complexes (COCs) in 2-Day and 3-Day group, respectively. Significantly higher proportion of expanded oocytes was recovered in 3-Day protocol compared to 2-Day protocol (36.0% vs. 13.2%) and higher number of compact COCs were recovered in 2-Day protocol. In 3-Day protocol, significantly higher percentage of oocytes retrieved was at Metaphase-II stage compared to 2-Day protocol (40% vs.15.8%, respectively). The preliminary trial demonstrated that 3-day superstimulatory protocol yielded higher percentage of in vivo matured oocytes than 2-Day protocol in buffaloes.

Design and fabrication of aspiration microfluidic channel for oocyte characterization

The development potential for oocytes can be predicted by their mechanical properties. One important parameter that is measured to calculate oocyte hardness is Cortical Tension (CT). In this work, for the first time, we present the design, simulation, and fabrication of a new aspiration microfluidic chip to measure the CT of oocytes and then predict their maturation capability in the Germinal Vesicle (GV) stage. This high-performance technique facilitates oocyte characterization and is a promising alternative to traditional methods such as MicroPipette Aspiration (MPA). The proposed technique involves considerably simpler operation, less specialized equipment, and less technical skill than MPA. The proposed microfluidic channel also promises faster measurements. It is shown that in order to completely continue the growth process of oocytes in GV stage, the CT should be in a certain range: very low or very high CTs lead to unsuccessful growth. The obtained results show that 79% of oocytes with the CT between 1.5 and 3 nN/μm reach the Metaphase II (MII) stage, whereas the growth for 78% of oocytes with the CT less than 1.5 nN/μm or higher than 3 nN/μm stops at the GV or Germinal Vesicle Break Down (GVBD) stages. Another property, kvis, that points to the viscous behavior of oocytes is also measured. It is seen that 80% of GV oocytes with the kvis values between 15 and 30 k Pa s/m reach the MII stage.

Expression patterns and biological function of BCL2L10 during mouse preimplantation development

BCL2-like 10 (BCL2L10) is abundantly expressed in mammalian oocytes and plays a crucial role in the completion of oocyte meiosis. However, the expression patterns of BCL2L10 and its biological functions during preimplantation development have not been well characterized. Here, we investigated the spatiotemporal expressions of Bcl2l10 during mouse preimplantation development using RT-qPCR and immunofluorescence and its biological function using siRNA and morpholino injection into pronuclear embryos. Results from RT-qPCR showed that Bcl2l10 was highly expressed in the metaphase Ⅱ-stage oocytes and pronuclear-stage embryos, but expression markedly decreased from the two-cell stage onwards and was no longer detected at the four-cell stage and beyond. Immunofluorescence staining showed that BCL2L10 was detectable throughout preimplantation development and localized in the cytoplasm and nuclei. Knocking down Bcl2l10 resulted in a reduced blastocyst formation rate (P < 0.01) and decreased expression of OCT4, NANOG, and SOX17 (P < 0.05). We concluded that the role of BCL2L10 is strongly associated with developmental competence of preimplantation mouse embryos.

Proteomic analysis implicates that postovulatory aging leads to aberrant gene expression, biosynthesis, RNA metabolism and cell cycle in mouse oocytes

BackgroundIn mammals, oocytes display compromised quality after experiencing a process of postovulatory aging. However, the mechanisms underlying are not yet fully understood. Here, we portrayed a protein expression profile of fresh and aging metaphase II (MII) mouse oocytes by means of four-dimensional label-free quantification mass spectrometry (4D-LFQ).ResultsThe analysis of 4D-LFQ data illustrated that there were seventy-six differentially expressed proteins (DEPs) between two groups of MII stage oocytes. Fifty-three DEPs were up-regulated while twenty-three DEPs were down-regulated in the MII oocytes of the aging group, and Gene Ontology (GO) analysis revealed that these DEPs were mainly enriched in regulation of gene expression, biosynthesis, RNA metabolism and cell cycle. Our detailed analysis revealed that the expression of proteins that related to gene expression processes such as transcription, translation, post-translational modifications and epigenome was changed; the relative protein expression of RNA metabolic processes, such as RNA alternative splicing, RNA export from nucleus and negative regulation of transcription from RNA polymerase II promoter was also altered.ConclusionIn conclusion, we identified considerable DEPs and discussed how they agreed with previous researches illustrating altered protein expression associated with the quality of oocytes. Our research provided a new perspective on the mechanisms of postovulatory aging and established a theoretical support for practical methods to control and reverse postovulatory aging.

Cilostamide, a phosphodiesterase 3A inhibitor, sustains meiotic arrest of rat oocytes by modulating cyclic adenosine monophosphate level and the key regulators of maturation promoting factor.

Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.

Rescue IVM of Denuded GV- and MI-Stage Oocytes of Premenopausal Rats with Oncostatin M, Insulin-like Growth Factor I, and Growth Hormone.

Immature oocytes are retrieved and matured through in vitro maturation (IVM). Maturation, fertilization rates, and embryo development via IVM are all lower than those found in vitro fertilization (IVF) cycles. We investigated the effects of oncostatin M (OSM), insulin-like growth factor-1 (IGF-I), and growth hormone (GH) in rescue IVM. A total of 111 germinal vesicle (GV) and 17 metaphase I (MI) oocytes were obtained after conventional IVF from 28 female Wistar albino rats. Denuded immature oocytes were cultured in maturation medium supplemented with OSM, IGF-1, or GH. The quantities of metaphase II (MII) oocytes matured from the GV stage were 17 of 30 (56.6%), 15 of 28 (53.5%), 10 of 30 (33.3%), and 7 of 23 (30.3%), in control, OSM, IGF-I, and GH groups, respectively. Maturation rates in control and OSM groups were higher than those in IGF-I and GH groups (p = 0.001). The quantities of MII oocytes matured from MI stage were 7 of 7 (100%), 4 of 4 (100%), 1 of 1 (100%), and 1 of 5 (20%) in control, OSM, IGF-I, and GH groups, respectively. Maturation rates from MI to MII stages in control, OSM, and IGF-I groups were higher than those in the GH group (p = 0.004). Acceptable maturation rates are observed with OSM in rat oocytes in rescue IVM.