Cortical Granule Release Research Articles

Dithiothreitol prevents the spontaneous release of cortical granules in in vitro aged mouse oocytes by protecting regulatory proteins of cortical granules exocytosis and thickening the cortical actin cytoskeleton

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes.

Chronological age enhances aging phenomena and protein nitration in oocyte.

The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging. Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration. At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition. Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease.

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)-dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

Zinc Protection of Fertilized Eggs is Conserved in Non-Mammalian Species

Polyspermy, the fertilization of an egg by multiple sperm, is embryonic lethal in most sexually reproducing species. As such, most eggs utilize strategies to protect zygotes from further fertilizations. One of these mechanisms is the slow block to polyspermy, which involves the release of cortical granules from the egg. This exocytosis event modifies the extracellular matrix of the eggs to become impermeable to sperm. In mammals, zinc is released as part of the slow block and is hypothesized to contribute to inhibition of fertilization. We demonstrate that zinc release also occurs during activation of eggs in the frog Xenopus laevis, the axolotl Ambystoma mexicanum, and the fish Danio rerio. By assaying for embryonic development from eggs inseminated in varying concentrations of zinc, we found that zinc inhibits fertilization in X. laevis, as well as the sea urchin Strongylocentrotus purpuratus, and in cnidarian Hydractinia symbiolonigarpus. In X. laevis, inhibition of fertilization by various transition metals suggests that zinc coordinates proteins in the extracellular matrix to prevent sperm penetration. Also in X. laevis, whole cell recording data from eggs treated with zinc suggests that the cation slowly depolarizes the cell. Our data represents evidence of zinc release acting as a well conserved strategy to prevent supernumerary sperm entry in eggs of sexually reproducing organisms from mammals to cnidarians.

Effect and possible mechanisms of melatonin treatment on the quality and developmental potential of aged bovine oocytes.

After reaching the metaphase II (MII) stage, unfertilised oocytes undergo a time-dependent process of quality deterioration referred to as oocyte aging. The associated morphological and cellular changes lead to decreased oocyte developmental potential. This study investigated the effect of exogenous melatonin supplementation on in vitro aged bovine oocytes and explored its underlying mechanisms. The levels of cytoplasmic reactive oxygen species and DNA damage response in bovine oocytes increased during in vitro aging. Meanwhile, maturation promoting factor activity significantly decreased and the proportion of morphologically abnormal oocytes significantly increased. Melatonin supplementation significantly decreased quality deterioration in aged bovine MII oocytes (P<0.05). Additionally, it decreased the frequency of aberrant spindle organisation and cortical granule release during oocyte aging (P<0.05). In the melatonin-supplemented group, mitochondrial membrane potential and ATP production were significantly increased compared with control. Furthermore, melatonin treatment significantly increased the speed of development of bovine oocytes to the blastocyst stage after in vitro fertilisation and significantly decreased the apoptotic rate in the blastocysts (P<0.05). The expression of Bax and Casp3 in the blastocysts was significantly reduced after treatment with melatonin, whereas expression of Bcl2 significantly increased (P<0.05). In conclusion, these findings suggest that supplementation of aged bovine oocytes with exogenous melatonin improves oocyte quality, thereby enhancing the developmental capacity of early embryos.

Post-ovulatory aging of mouse oocytes in vivo and in vitro: Effects of caffeine on exocytosis and translocation of cortical granules.

The developmental potential of post-ovulatory oocytes decreases with aging in vivo and in vitro. In this study, we aimed to investigate the effects of a potent antioxidant caffeine on cortical granules (CGs) distribution in mouse oocytes aging in vivo and in vitro. We found that in vivo administration of 150 mg/kg caffeine caused ovulation of some morphologically abnormal oocytes showing premature exocytosis or congregation of CGs, but significantly decreased abnormal distribution of CGs in oocytes aging for 6 h, 12 h and 18 h in vivo compared to those without caffeine treatment. Unexpectedly, supplementation of oocyte culture medium with 10 mmol/L caffeine accelerated CGs release of oocytes and the normal CG distribution rate dramatically decreased from 6 h in oocytes aging in vitro. It appeared that oocytes showed a high degree of abnormal CG distribution by aging for 18 h, and caffeine might delay oocyte CG exocytosis in vivo, but accelerates CG exocytosis in vitro. Our findings may have implications for improving assisted reproduction technologies.

Human oocyte developmental potential is predicted by mechanical properties within hours after fertilization.

The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage.

Aura (mid1ip1l) regulates the cytoskeleton at the zebrafish egg-to-embryo transition.

Embryos from females homozygous for a recessive maternal-effect mutation in the gene aura exhibit defects including reduced cortical integrity, defective cortical granule (CG) release upon egg activation, failure to complete cytokinesis, and abnormal cell wound healing. We show that the cytokinesis defects are associated with aberrant cytoskeletal reorganization during furrow maturation, including abnormal F-actin enrichment and microtubule reorganization. Cortical F-actin prior to furrow formation fails to exhibit a normal transition into F-actin-rich arcs, and drug inhibition is consistent with aura function promoting F-actin polymerization and/or stabilization. In mutants, components of exocytic and endocytic vesicles, such as Vamp2, Clathrin and Dynamin, are sequestered in unreleased CGs, indicating a need for CG recycling in the normal redistribution of these factors. However, the exocytic targeting factor Rab11 is recruited to the furrow plane normally at the tip of bundling microtubules, suggesting an alternative anchoring mechanism independent of membrane recycling. A positional cloning approach indicates that the mutation in aura is associated with a truncation of Mid1 interacting protein 1 like (Mid1ip1l), previously identified as an interactor of the X-linked Opitz G/BBB syndrome gene product Mid1. A Cas9/CRISPR-induced mutant allele in mid1ip1l fails to complement the originally isolated aura maternal-effect mutation, confirming gene assignment. Mid1ip1l protein localizes to cortical F-actin aggregates, consistent with a direct role in cytoskeletal regulation. Our studies indicate that maternally provided aura (mid1ip1l) acts during the reorganization of the cytoskeleton at the egg-to-embryo transition and highlight the importance of cytoskeletal dynamics and membrane recycling during this developmental period.

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes.

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts.

Zinc depletion activates porcine metaphase II oocytes independently of the protein kinase C pathway.

Zinc is an important trace element that regulates several biological functions. This study investigated the role of zinc in the metaphase (M) II arrest of porcine oocytes. N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn(2+) chelator, was used to deplete free zinc from porcine MII stage oocytes. TPEN treatment significantly (P < 0.01) reduced the zinc content in the cytoplasm. The percentages of oocytes in which second polar body emission and pronuclear formation occurred increased as the concentration of TPEN increased (P < 0.01), and reached 93.64 ± 5.53% and 90.61 ± 9.10%, respectively, following treatment with 10 μM TPEN. Zinc depletion also resulted in cortical granule release and spindle depolarization. Maturation-promoting factor activity, as assessed by examining p34(cdc2) activity, decreased (P < 0.05) following zinc depletion. Following TPEN treatment, embryos developed to the 4-cell stage but failed to reach the blastocyst stage. Zinc release is a common event in protein kinase C (PKC) activation. Therefore, we examined the impact of zinc depletion on phosphorylation of PKC substrates. Phosphorylation of PKC substrates was reduced (P < 0.05) in zinc-depleted oocytes, and this was rescued by phorbol 12-myristate 13-acetate (PMA) treatment. However, treatment of oocytes with both PMA and TPEN did not affect pronuclear formation or second polar body emission. These data are inconsistent with the hypothesis that oocyte activation caused by zinc depletion is mediated by the PKC pathway. This study shows that zinc has a novel role in maintaining MII arrest in porcine oocytes, but this is not mediated by the PKC pathway.

Vitrification of immaturemouse oocytes by the modified‐cut standard straw method

The feasibility of using the modified-cut standard straw (M-CSS) method for the vitrification of immature mouse oocytes has been tested. The effects of different vitrification methods on oocyte survival, cytoskeletal organization, the distribution of cortical granules (CGs), and apoptosis have also been compared. Immature mouse oocytes were vitrified-thawed using electron microscope grid or M-CSS method, and cultured to meiosis II (MII) stage. Oocyte development, cytoskeletal organization, CG distribution, and the expression of apoptosis-related genes were evaluated. Rates of recovery (91.7 vs. 74.9%) and survival (89.0 vs. 62.6%) were significantly higher in M-CSS group than in EM grid group. The number of oocytes with normal chromosome alignment at the spindle and spindle morphology were similar in both groups. However, the actin cap was significantly degraded in EM grid groups (52.6 vs. 35.1%, respectively). Abnormal release of CGs also frequently occurred in EM grid groups (42.6 vs. 32.7%, respectively). Pro-apoptosis-related gene expression levels of Bax, caspase 3 were expressed lower than control in MII stage oocytes derived from M-CSS group; anti apoptosis-related genes, survivin and heat shock factor-1 (Hsf-1) were slightly increased. However, all genes expression was significantly increased in MII stage oocytes derived from EM grid groups. Vitrification reduces the survival rate of immature mouse oocytes, alters cytoskeletal organization and CG distribution, and promotes apoptosis. However, these effects are less pronounced in vitrified oocytes generated by M-CSS than in those generated by EM grid method. Therefore, the novel M-CSS is a feasible approach for the cryopreservation of immature mouse oocytes.

Maturation conditions and boar affect timing of cortical reaction in porcine oocytes

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.

182 CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE OOCYTES PROVOKES FULL CORTICAL REACTION: AN APPROACH TO STUDY CORTICAL GRANULE-DERIVED PROTEINS IN PIGS

The cortical reaction is a mechanism that prevents polyspermy by cortical granule content being released into the periviteline space, modifying the zona pellucida (ZP). Knowledge about specific cortical granule-derived proteins has progressed slowly because these organelles contain only picogram quantities of proteins. An efficient method for collecting cortical granule content would help in its study; chemical activation of ZP-free oocytes has been successfully used in the murine model (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Calcium ionophore A23187 is an effective chemical stimulator for provoking the cortical reaction in ZP-intact pig oocytes. However, the commonly used protocol (50 μM for 5min) cannot be employed with ZP-free oocytes because the oolemma is damaged, oocyte lysed and medium contaminated with ooplasm content, which is necessary to reduce the time and ionophore concentration (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst). The objective of this study was to evaluate the efficiency of this activation protocol for provoking the cortical reaction in ZP-free oocytes by assessment with confocal and electron microscopy. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with pronase. Then, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM for 2min), transferred to an exudate medium and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. Control ZP-free oocytes were incubated without being activated. After incubation, ionophore-treated (n = 10) and control oocytes (n = 18) were used to assess the presence of a cortical granule monolayer. An aliquot was fixed, permeabilized (0.1% Triton), incubated with peanut agglutinin lectin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min) and examined under a confocal microscope. Presence or absence of a cortical granule monolayer at the equator level was recorded. Another aliquot was fixed and processed for electron microscopy observation. The cortical granules in the whole oocytes were counted and results are presented as the mean ± standard error of the mean. No cell lysis was observed in control or activated ZP-free oocytes after treatment and incubation time. The confocal study showed that the activation protocol provokes a full cortical reaction in 100% of A23187-treated oocytes, given that no peanut agglutinin labeling was observed in the cortical area. Presence of a cortical granule monolayer under the oolemma was observed in 100% of control oocytes. Cortical granule release was confirmed by electron microscopy. Control oocytes had 5.90 ± 1.78 cortical granules per 5 μm of oolemma, whereas activated oocytes exhibited a significant reduction (P &lt; 0.05) of up to 0.71 ± 0.20. In conclusion, the presented activation protocol by using ZP-free oocytes is a valid method for provoking a complete cortical reaction and could be employed in the future as an efficient method to collect cortical granule-derived proteins in pig oocytes. Supported by CONACYT (0105961/I0110/194/09), MEC and FEDER (AGL2009-12512-C02-01).

Morphology of meiotic spindles, chromosomes, microtubules and cortical granules in vitrified human metaphase II oocytes after thawing

Exposure of metaphase II (MII) mammal oocytes to cryopreservation process has been reported to cause disorganization of meiotic spindles (MS) and premature release of cortical granules (CG) without sperm penetration. The present study was conducted to observe morphology of vitrified human MII oocytes chronologically by polarization microscopy (Polscope), fluorescent microscopy and transmission electron microscopy (TEM) after thawing for determination of the best timing for ICSI.

Calmodulin-dependent protein kinase gamma 3 (CamKIIγ3) mediates the cell cycle resumption of metaphase II eggs in mouse

Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca(2+) signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIgamma3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca(2+) stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIgamma3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIgamma3 plays a necessary and sufficient role in transducing the oscillatory Ca(2+) signal into cell cycle resumption, but not into cortical granule release.

O-154: The effect of chelating cytosolic calcium on the viability of vitrified mouse oocytes

Vitrification is an alternative to cryopreserve MII oocytes, however, the embryonic potential of oocytes vitrified by choline chloride DMSO-EG-sucrose vitrification system is still low. Recently, it has been reported that DMSO and EG may induce the increase of intracellular calcium. This increase in calcium may have detrimental effects: premature cortical granule release, zona pellucida hardening, metaphase-to-anaphase transition, and/or egg activation. Preventing the increase of intracellular calcium by calcium-free media combined with a cell membrane permeable calcium chelator was carried out to assess the viability of mouse oocytes exposed to a choline chloride DMSO-EG-sucrose based vitrification system. Prospective study using mouse oocytes at a University-based research laboratory. MII oocytes were collected from 6-8 weeks old CB6F1 mice by hyperstimulation of 5IU PMSG and 5IU hCG. MII oocytes were denuded by brief exposure to 100iu/ml hyaluronidase, and then cultured in vitro less than 1hour prior to exposure of choline chloride DMSO-EG-sucrose vitrification system. Oocytes were loaded in pulled straw and heat-sealed just before plunging directly into liquid nitrogen. The experimental groups include presence or absence of calcium in the vitrification system, and concentration of BAPTA AM, a cell permeant calcium chelator. Group 1, calcium-based PBS; Group 2: calcium-free PBS; Groups 3 - 7: calcium-free PBS + varying concentrations (0.1μM, 0.5μM, 5μM, 50μM and 500μM) of BAPTA AM. After thawing, eggs were allowed to recover for one hour. Surviving oocytes were inseminated by capacitated sperm. Embryos were cultured in sequential Quinn’s Media (Sage) supplemented with 6% plasmanate, and monitored at day 1 and day 5(Table 1). Day 5 blastocysts were graded and corroborated by double staining of Oct 3/4 (detect functional inner cell mass) and of Hoechst 33342 (detect total embryonic cell number). Mouse oocytes, in response to 0.1μM BAPTA based vitrification system, display high rate of survival (97%) and cleavage (71%), comparable to that response to calcium based and calcium-free based vitrification system. However, raising the concentration of BAPTA to 0.5μM and above, the rate of survival (<82%) and cleavage (<55%) declined dramatically. Furthermore, more embryos in group of 0.1μM BAPTA develop into good (45% ≥3BB) and excellent (32% ≥4AA) blastocyst than in other groups. The group of 0.5μM BAPTA yielded less blastocysts, but still generated a number of good and excellent blastocysts comparable to the group of 0.1μM BAPTA. BAPTA influences the viability of mouse oocytes in a concentration dependant manner. BAPTA, at a concentration of 0.1μM (equivalent to cytosolic Ca2+ concentration -10-7 M = 0.1μM), exhibits the maximum benefit to improve the viability of vitrified oocytes. The effect of BAPTA on post-implantation embryos and full-term offspring needs to be addressed in further study.

Cortical granules behave differently in mouse oocytes matured under different conditions

To better understand the differences between in vivo (IVO) and in vitro (IVM) matured oocytes, we studied the chronological changes in cortical granule (CG) distribution and nuclear progression during maturation, and the competence of CG release and embryo development of mouse oocytes matured under different conditions. Oocytes matured in vivo or in different culture media were used and CG distribution and release were assessed by fluorescein isothiocyanate-labelled Lens culinaris agglutinin and laser confocal microscopy. Tempos of nuclear maturation and CG redistribution were slower, and competence for CG exocytosis, cleavage and blastulation were lower in the IVM oocytes than in the IVO oocytes. These parameters also differed among oocytes matured in different culture media. Hypoxanthine (HX, 4 mM) blocked germinal vesicle breakdown (GVBD), postponed CG migration and prevented CG-free domain (CGFD) formation. Cycloheximide (CHX) facilitated both GVBD and CG migration, but inhibited CGFD formation. The presence of serum in maturation media enhanced CG release after aging or activation of oocytes. Maintenance of germinal vesicle intact for some time by a trace amount (0.18 mM) of HX was beneficial to oocyte cytoplasmic maturation. CGs behaved differently in mouse oocytes matured under different conditions, and cytoplasmic maturity was not fully achieved in the IVM oocytes.

Zona Pellucida Modifications and Fertilization Following Metaphase II Oocyte Vitrification and Warming

Oocyte cryopreservation holds promise for preserving fertility in women undergoing cancer therapies, and can be achieved by slow-rate freezing or vitrification. Oocyte slow-rate freezing compromises zona pellucida (ZP) function, necessitating insemination/fertilization by intracytoplasmic sperm injection (ICSI). Whether oocyte vitrification causes similar ZP modifications is unknown. Objectives were to evaluate effects of vitrification/warming on oocyte fertilization by conventional in vitro insemination, ZP solubility, cortical granule (CG) release, fertilization by ICSI, and ability of fetuin to prevent ZP modifications.

Cumulus Cells Accelerate Aging of Mouse Oocytes1

The role of cumulus cells (CCs) that surround oocytes in maturation, ovulation, and fertilization has been extensively studied, yet little is known about their role in oocyte aging. Although early studies have shown that when ovulated oocytes are aged in vitro displayed similar morphological alterations as those aged in vivo, a recent study found that vitro culture of mouse oocytes retarded oocyte aging. The objective of this study was to test the hypothesis that CCs would accelerate oocyte aging. During in vitro aging with CCs of both in vivo-matured and in vitro-matured mouse oocytes, activation rates increased, whereas the maturation-promoting factor (MPF) activity decreased significantly as during in vivo aging of the ovulated oocytes. During aging after denudation of CCs, however, activation rates of both in vivo-matured and in vitro-matured oocytes remained low and the MPF activity decreased much more slowly compared to that of oocytes aged with CCs. Although many oocytes aged in vivo and in vitro with CCs showed a partial cortical granule (CG) release, very few cumulus-free oocytes released their CGs during in vitro aging. When denuded oocytes were cultured with cumulus-oocyte-complexes at a 1:2 ratio or on a CC monolayer, activation rates increased, while MPF activity decreased significantly. The results strongly suggested that CCs accelerated the aging progression of both in vivo-matured and in vitro-matured mouse oocytes.

Attribution of cortical granules to formation of fertilization envelopes and polyspermy block inurechis unicinctus

Abstract Cortical reaction and polyspermy block are well defined in most marine invertebrates. In Urechis species, the function of cortical granules (CGs) is not yet known, and there is controversy on whether the cortical reaction occurs, or the fertilization envelope (FE) is attributed to CG releases or functions to prevent polyspermy. This study was carried out to determine the cortical reactions and functions of the FE in Urechis unicinctus. Artificial insemination of the eggs revealed that CG release occurred to give rise to perivitelline space (PS) into the final FE. Both PS and final FE effectively blocked polyspermy. The final FE was accomplished within 10 min after sperm‐egg initial binding. No massive release of CGs occurred within the early phase of 5 min after the initial binding, initially and the PS seemed to play a role to prevent polyspermy. The CG massively released its content into the PS in late phase of FE formation, and differentiated PS into five intermediate layers. The layers opened...