Hyo J. Lee

Cortical Mechanics and Meiosis II Completion in Mammalian Oocytes Are Mediated by Myosin-II and Ezrin-Radixin-Moesin (ERM) Proteins

Analysis of mouse oocyte mechanics shows that effective tension drops 6-fold from prophase I to metaphase II; the metaphase II egg has a 2.5-fold tension differential between the cortex over the spindle and the opposite cortex. Manipulation of actin, myosin-II, or ERMs alters tension levels and induces spindle abnormalities during meiosis II.

Prophase I Mouse Oocytes Are Deficient in the Ability to Respond to Fertilization by Decreasing Membrane Receptivity to Sperm and Establishing a Membrane Block to Polyspermy

ABSTRACT Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm.

MAPK3/1 (ERK1/2) and Myosin Light Chain Kinase in Mammalian Eggs Affect Myosin-II Function and Regulate the Metaphase II State in a Calcium- and Zinc-Dependent Manner

ABSTRACT Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

Cortical mechanics and myosin-II abnormalities associated with post-ovulatory aging: Implications for functional defects in aged eggs

STUDY HYPOTHESIS Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovulated mouse eggs were collected at 21-22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13-14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. LIMITATIONS, REASONS FOR CAUTION These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. WIDER IMPLICATIONS OF THE FINDINGS These data provide insights into causes of reproductive failures at later post-copulatory times. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest.

Effects of inhibition of Ubiquitin C‐Terminal Hydrolase L1 (UCH‐L1) on sperm incorporation and cortical tension in mouse eggs

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) is thought to have multiple functions in mammalian oocytes and early embryos (e.g., Mtango et al., 2011; Sekiguchi et al., 2006). Here we tested the overall hypothesis that UCH-L1 has functions in the mouse egg cortex and/or overlying plasma membrane, given that UCH-L1 is enriched in the egg cortex (Sekiguchi et al., 2006). We examined egg membrane receptivity to sperm by assessing sperm incorporation over time with zona pellucida-free eggs, as previously described (McAvey et al., 2002), in the presence or absence of the UCH-L1 inhibitor LDN-57444, the development and specificity of which was described by Liu et al. (2003). Zona-pellucida-free eggs from superovulated CF-1 female mice were treated with 0, 5, or 10mM LDN-57444 (Calbiochem, San Diego, CA; stock solution prepared in DMSO) for 2 hr, inseminated with 10 sperm/ml, and then examined at 1 and 3 hr post-insemination. Under these conditions, control eggs are fertilized effectively and polyspermy is low (Table 1). In contrast, LDN57444-treated eggs show an increase in polyspermy and in sperm-egg fusion events over time (Table 1). This suggested that inhibition of UCH-L1 disrupts the ability of the egg membrane to convert to an unreceptive state after fertilization. The membrane block to polyspermy is mediated in part by the egg cytoskeleton, and experimental manipulations that disrupt establishment of thismembraneblock also alter tension in the cortical cytoskeleton (McAvey et al., 2002; Larson et al., 2010). We therefore assessed the effects of LDN-57444 on cortical tension in eggs using micropipette aspiration (Larson et al., 2010), a highly sensitive readout of contractility in the cortical cytoskeleton that is regulated by actin, non-muscle myosin-II, and links between actin filaments and to theoverlyingplasmamembrane. LDN-57444treated eggs had decreased cortical tension as compared to DMSO-treated eggs, with a nearly 50% decrease in the microvillar domain, which supports sperm binding and fusion, and the amicrovillar domain, which sequesters the meiotic spindle (Table 1). Together, these data indicate that UCH-L1 affects certain features of the egg cortex and plasma membrane. The ubiqutitin system in general, and UCH-L1 specifically, has been implicated in the function of actinassociated processes and structures, including cortical tension (e.g., Bass eres et al., 2010; Vergara et al., 2014). The data here support a model that the egg cortex in some way impacts the egg’s responsiveness to a fertilizing sperm and/or conversion of the membrane to an unreceptive state after the first sperm has penetrated (McAvey et al., 2002). Although increased polyspermy was not detected in in vivo fertilization studies with one mouse model with a Uchl1 mutation (Mtango et al., 2011), increased polyspermy was observed following in vitro fertilization of eggs from this mutant model (Sekiguchi et al., 2006), which is consistent with the fact that multiple factors contribute to polyspermy prevention in vivo. The assays used here, including acute pharmacological inhibition of UCH-L1 activity, complement the in vivo, genetic studies using models with chronic UCH-L1 deficiency, and support the use of combined approaches to advance our understanding of reproductive defects. In particular, elucidating factors that contribute to polyspermic fertilization has high relevance to human reproductive health, given that dispermic fertilization occurs in human conceptions in vivo and in vitro (e.g., Zaragoza et al., 2000).

Effects of Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) inhibition on sperm incorporation and cortical tension in mouse eggs.

Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) is thought to have multiple functions in mammalian oocytes and early embryos (e.g., Mtango et al., 2011; Sekiguchi et al., 2006). Here we tested the overall hypothesis that UCH-L1 has functions in the mouse egg cortex and/or overlying plasma membrane, given that UCH-L1 is enriched in the egg cortex (Sekiguchi et al., 2006). We examined egg membrane receptivity to sperm by assessing sperm incorporation over time with zona pellucida-free eggs, as previously described (McAvey et al., 2002), in the presence or absence of the UCH-L1 inhibitor LDN-57444, the development and specificity of which was described by Liu et al. (2003). Zona-pellucida-free eggs from superovulated CF-1 female mice were treated with 0, 5, or 10mM LDN-57444 (Calbiochem, San Diego, CA; stock solution prepared in DMSO) for 2 hr, inseminated with 10 sperm/ml, and then examined at 1 and 3 hr post-insemination. Under these conditions, control eggs are fertilized effectively and polyspermy is low (Table 1). In contrast, LDN57444-treated eggs show an increase in polyspermy and in sperm-egg fusion events over time (Table 1). This suggested that inhibition of UCH-L1 disrupts the ability of the egg membrane to convert to an unreceptive state after fertilization. The membrane block to polyspermy is mediated in part by the egg cytoskeleton, and experimental manipulations that disrupt establishment of thismembraneblock also alter tension in the cortical cytoskeleton (McAvey et al., 2002; Larson et al., 2010). We therefore assessed the effects of LDN-57444 on cortical tension in eggs using micropipette aspiration (Larson et al., 2010), a highly sensitive readout of contractility in the cortical cytoskeleton that is regulated by actin, non-muscle myosin-II, and links between actin filaments and to theoverlyingplasmamembrane. LDN-57444treated eggs had decreased cortical tension as compared to DMSO-treated eggs, with a nearly 50% decrease in the microvillar domain, which supports sperm binding and fusion, and the amicrovillar domain, which sequesters the meiotic spindle (Table 1). Together, these data indicate that UCH-L1 affects certain features of the egg cortex and plasma membrane. The ubiqutitin system in general, and UCH-L1 specifically, has been implicated in the function of actinassociated processes and structures, including cortical tension (e.g., Bass eres et al., 2010; Vergara et al., 2014). The data here support a model that the egg cortex in some way impacts the egg’s responsiveness to a fertilizing sperm and/or conversion of the membrane to an unreceptive state after the first sperm has penetrated (McAvey et al., 2002). Although increased polyspermy was not detected in in vivo fertilization studies with one mouse model with a Uchl1 mutation (Mtango et al., 2011), increased polyspermy was observed following in vitro fertilization of eggs from this mutant model (Sekiguchi et al., 2006), which is consistent with the fact that multiple factors contribute to polyspermy prevention in vivo. The assays used here, including acute pharmacological inhibition of UCH-L1 activity, complement the in vivo, genetic studies using models with chronic UCH-L1 deficiency, and support the use of combined approaches to advance our understanding of reproductive defects. In particular, elucidating factors that contribute to polyspermic fertilization has high relevance to human reproductive health, given that dispermic fertilization occurs in human conceptions in vivo and in vitro (e.g., Zaragoza et al., 2000).