Effects of Dithiothreitol on Fertilization and Early Development in Sea Urchin

The vitelline layer (VL) of a sea urchin egg is an intricate meshwork of glycoproteins that intimately ensheathes the plasma membrane. During fertilization, the VL plays important roles. Firstly, the receptors for sperm reside on the VL. Secondly, following cortical granule exocytosis, the VL is elevated and transformed into the fertilization envelope (FE), owing to the assembly and crosslinking of the extruded materials. As these two crucial stages involve the VL, its alteration was expected to affect the fertilization process. In the present study, we addressed this question by mildly treating the eggs with a reducing agent, dithiothreitol (DTT). A brief pretreatment with DTT resulted in partial disruption of the VL, as judged by electron microscopy and by a novel fluorescent polyamine probe that selectively labelled the VL. The DTT-pretreated eggs did not elevate the FE but were mostly monospermic at fertilization. These eggs also manifested certain anomalies at fertilization: (i) compromised Ca2+ signaling, (ii) blocked translocation of cortical actin filaments, and (iii) impaired cleavage. Some of these phenotypic changes were reversed by restoring the DTT-exposed eggs in normal seawater prior to fertilization. Our findings suggest that the FE is not the decisive factor preventing polyspermy and that the integrity of the VL is nonetheless crucial to the egg’s fertilization response.

Knockin’ on Egg’s Door: Maternal Control of Egg Activation That Influences Cortical Granule Exocytosis in Animal Species

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

PSV-10 Effects of l-α-amino Butyrate Supplementation on Oocyte Maturation

L-α-amino butyrate is a low-molecular weight thiol compound that acts to increase the levels of glutathione in the oocyte. Glutathione acts as an antioxidant during oocyte maturation and promotes male pronuclear formation during fertilization. Supplementing the L-α-amino butyrate helps to decrease polyspermic penetration rates and improve early embryonic development in swine. However, it is unknown if L-α-amino butyrate supplementation affects the environment of the oocyte or the oocyte directly. Therefore, the objective of this study was to determine if L-α-amino butyrate supplementation to the maturation media acted on the oocyte or had alternative beneficial effects in the surrounding environment. Oocytes were randomly assigned to a maturation media containing an amino acid transport inhibitor, quisqualic acid (QA) (0 or 1 mM) and then supplemented with L-α-amino butyrate (0 or 3.3 mM). Oocytes were evaluated for stage of meiosis (n=380) and cumulus cell expansion (n=411) at the end of maturation. The remaining oocytes were fertilized and evaluated for cortical granule exocytosis (n=400) and IVF kinetics (n=456). Supplementation of L-α-amino butyrate with or without QA significantly increased (P < 0.05) cumulus cell expansion, cortical granule exocytosis and male pronuclear formation compared to no supplementation or QA supplementation. There was no difference in meiotic progression, fertilization or polyspermic penetration rates between the treatment groups. Results suggest that when L-α-amino butyrate is supplemented during maturation, it improves the maturation of the oocyte by acting directly on the oocyte and not through the surrounding environment of the oocyte.

PSV-11 Efficacy of Quisqualic Acid as an Amino Acid Transporter Inhibitor in Pig Oocytes

Quisqualic acid is a known inhibitor of sodium-dependent amino acid transporters. However, it is unknown if quisqualic acid has similar effects in in vitro mature oocytes. Therefore, the objective of this study was to determine the optimal dose and effects of quisqualic acid supplemented during maturation. Oocytes (n=362) were supplemented during maturation with quisqualic acid (0, 0.5, 0.75, 1.0, 2.5 mM) to determine the minimum concentration of quisqualic acid that had no effect on oocyte maturation but significantly decreased the intracellular glutathione concentration. The addition of 1.0 mM quisqualic acid was the lowest concentration observed to cause intracellular glutathione levels to be significantly less (P < 0.05) without affecting maturation compared to no quisqualic acid. Based on those results, oocytes were supplemented with or without 1.0 mM quisqualic acid then evaluated for cumulus cell expansion (n=410) and stage of meiosis (n=380) at the end of maturation. Additional oocytes were fertilized and assessed for cortical granule exocytosis (n=400) and kinetics at 12 h after IVF (n=420). Supplementing quisqualic acid to the media did not have an effect on stage of meiosis, fertilization, polyspermic penetration, or cortical granule exocytosis. Supplementing 1.0 mM quisqualic acid significantly decreased (P < 0.05) cumulus cell expansion by the end of maturation and male pronuclear formation by 12 h after IVF. These results suggest that quisqualic acid supplementation during maturation in pigs inhibits sodium-dependent amino acid transporters.

Synaptotagmin 1 regulates cortical granule exocytosis during mouse oocyte activation

SummarySynaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

Pleiotropic effects of alpha-SNAP M105I mutation on oocyte biology: ultrastructural and cellular changes that adversely affect female fertility in mice

After sperm-oocyte fusion, cortical granules (CGs) located in oocyte cortex undergo exocytosis and their content is released into the perivitelline space to avoid polyspermy. Thus, cortical granule exocytosis (CGE) is a key process for fertilization success. We have demonstrated that alpha-SNAP -and its functional partner NSF- mediate fusion of CGs with the plasma membrane in mouse oocytes. Here, we examined at cellular and ultrastructural level oocytes from hyh (hydrocephalus with hop gait) mice, which present a missense mutation in the Napa gene that results in the substitution of methionine for isoleucine at position 105 (M105I) of alpha-SNAP. Mutated alpha-SNAP was mislocalized in hyh oocytes while NSF expression increased during oocyte maturation. Staining of CGs showed that 9.8% of hyh oocytes had abnormal localization of CGs and oval shape. Functional tests showed that CGE was impaired in hyh oocytes. Interestingly, in vitro fertilization assays showed a decreased fertilization rate for hyh oocytes. Furthermore, fertilized hyh oocytes presented an increased polyspermy rate compared to wild type ones. At ultrastructural level, hyh oocytes showed small mitochondria and a striking accumulation and secretion of degradative structures. Our findings demonstrate the negative effects of alpha-SNAP M105 mutation on oocyte biology and further confirm the relevance of alpha-SNAP in female fertility.

SNAP23 is required for constitutive and regulated exocytosis in mouse oocytes†

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using Trim-away, a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits regulated exocytosis in response to a Ca2+ stimulus (cortical granule exocytosis), as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs.