Oscillations in PP1 activity are essential for accurate progression through mammalian oocyte meiosis.

Tightly controlled fluctuations in kinase and phosphatase activity play important roles in regulating M-phase transitions. Protein Phosphatase 1 (PP1) is one of these phosphatases, with oscillations in PP1 activity driving mitotic M-phase. Evidence from a variety of experimental systems also points to roles in meiosis. Here, we report that PP1 is important for M-phase transitions through mouse oocyte meiosis. We employed a unique small-molecule approach to inhibit or activate PP1 at distinct phases of mouse oocyte meiosis. These studies show that temporal control of PP1 activity is essential for the G2/M transition, metaphase I/anaphase I transition, and the formation of a normal metaphase II oocyte. Our data also reveal that inappropriate activation of PP1 is more deleterious at the G2/M transition than at prometaphase I-to-metaphase I, and that an active pool of PP1 during prometaphase is vital for metaphase I/anaphase I transition and metaphase II chromosome alignment. Taken together, these results establish that loss of oscillations in PP1 activity causes a range of severe meiotic defects, pointing to essential roles for PP1 in female fertility, and more broadly, M-phase regulation.

Preventing polyspermy in mammalian eggs-Contributions of the membrane block and other mechanisms.

The egg's blocks to polyspermy (fertilization of an egg by more than one sperm) were originally identified in marine and aquatic species with external fertilization, but polyspermy matters in mammalian reproduction too. Embryonic triploidy is a noteworthy event associated with pregnancy complications and loss. Polyspermy is a major cause of triploidy with up to 80% of triploid conceptuses being the result of dispermic fertilization. The mammalian female reproductive tract regulates the number of sperm that reach the site of fertilization, but mammals also utilize egg-based blocks to polyspermy. The egg-based blocks occur on the mammalian egg coat (the zona pellucida) and the egg plasma membrane, with apparent variation between different mammalian species regarding the extent to which one or both are used. The zona pellucida block to polyspermy has some similarities to the slow block in water-dwelling species, but the mammalian membrane block to polyspermy differs substantially from the fast electrical block that has been characterized in marine and aquatic species. This review discusses what is known about the incidence of polyspermy in mammals and about the mammalian membrane block to polyspermy, as well as notes some lesser-characterized potential mechanisms contributing to polyspermy prevention in mammals.

Auxin-inducible protein degradation as a novel approach for protein depletion and reverse genetic discoveries in mammalian oocytes†.

The disruption of protein expression is a major approach used for investigating protein function in mammalian oocytes. This is often achieved with RNAi/morpholino-mediated knockdown or gene knockout, leading to long-term loss of proteins of interest. However, these methods have noteworthy limitations, including (a) slow protein turnover can prohibit use of these approaches; (b) essential roles in early events precludes characterization of functions in subsequent events; (c) extended protein loss can allow time for compensatory mechanisms and other unanticipated events that confound interpretation of results. The work presented here examines the use of auxin-inducible degradation, a powerful new approach that overcomes these limitations through the depletion of one's protein of interest through controllable ubiquitin-mediated degradation. This method has been employed in yeast and mammalian cell lines, and here we demonstrate the utility of auxin-inducible degradation in mouse oocytes at multiple stages of meiosis, through degradation of exogenously expressed EGFP. We also evaluate important parameters for experimental design for use of this system in oocytes. This study thus expands the toolkit of researchers in oocyte biology, establishing the use of this unique and versatile approach for depleting proteins in oocytes, and providing researchers with valuable information to make use of this system.

Micropipette Aspiration of Oocytes to Assess Cortical Tension.

Just as it is important to understand the cell biology of signaling pathways, it is valuable also to understand mechanical forces in cells. The field of mechanobiology has a rich history, including study of cellular mechanics during mitosis and meiosis in echinoderm oocytes and zygotes dating back to the 1930s. This chapter addresses the use of micropipette aspiration (MPA) to assess cellular mechanics, specifically cortical tension, in mammalian oocytes.

"Feeling the force" in reproduction: Mechanotransduction in reproductive processes.

Reproductive biologists are well-versed in many types of biochemical signaling, and indeed, there are almost innumerable examples in reproduction, including steroid and peptide hormone signaling, receptor-ligand and secondary messenger-mediated signaling, signaling regulated by membrane channels, and many others. Among reproductive scientists, a perhaps lesser-known but comparably important mode of signaling is mechanotransduction: the concept that cells can sense and respond to externally applied or internally generated mechanical forces. Given the cell shape changes and tissue morphogenesis events that are components of many phenomena in reproductive function, it should be no surprise that mechanotransduction has major impacts in reproductive health and pathophysiology. The conference on "Mechanotransduction in the Reproductive Tract" was a valuable launch pad to bring this hot issue in development, cell biology, biophysics, and tissue regeneration to the realm of reproductive biology. The goal of the meeting was to stimulate interest and increased mechanotransduction research in the reproductive field by presenting a broad spectrum of responses impacted by this process. The meeting highlighted the importance of convening expert investigators, students, fellows, and young investigators from a number of research areas resulting in cross-fertilization of ideas and suggested new avenues for study. The conference included talks on tissue engineering, stem cells, and several areas of reproductive biology, from uterus and cervix to the gametes. Specific reproductive health-relevant areas, including uterine fibroids, gestation and parturition, and breast tissue morphogenesis, received particular attention.

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.

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.

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.

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes.

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.

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.

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.

Sperm-egg interaction.

A crucial step of fertilization is the sperm-egg interaction that allows the two gametes to fuse and create the zygote. In the mouse, CD9 on the egg and IZUMO1 on the sperm stand out as critical players, as Cd9(-/-) and Izumo1(-/-) mice are healthy but infertile or severely subfertile due to defective sperm-egg interaction. Moreover, work on several nonmammalian organisms has identified some of the most intriguing candidates implicated in sperm-egg interaction. Understanding of gamete membrane interactions is advancing through characterization of in vivo and in vitro fertilization phenotypes, including insights from less robust phenotypes that highlight potential supporting (albeit not absolutely essential) players. An emerging theme is that there are varied roles for gamete molecules that participate in sperm-egg interactions. Such roles include not only functioning as fusogens, or as adhesion molecules for the opposite gamete, but also functioning through interactions in cis with other proteins to regulate membrane order and functionality.

The spatial and mechanical challenges of female meiosis.

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.

Membrane fusions during mammalian fertilization.

Successful completion of fertilization in mammals requires three different types of membrane fusion events. Firstly, the sperm cell will need to secrete its acrosome contents (acrosome exocytosis; also known as the acrosome reaction); this allows the sperm to penetrate the extracellular matrix of the oocyte (zona pellucida) and to reach the oocyte plasma membrane, the site of fertilization. Next the sperm cell will bind and fuse with the oocyte plasma membrane (also known as the oolemma), which is a different type of fusion in which two different cells fuse together. Finally, the fertilized oocyte needs to prevent polyspermic fertilization, or fertilization by more than one sperm. To this end, the oocyte secretes the contents of cortical granules by exocytotic fusions of these vesicles with the oocyte plasma membrane over the entire oocyte cell surface (also known as the cortical reaction or cortical granule exocytosis). The secreted cortical contents modify the zona pellucida, converting it to a state that is unreceptive to sperm, constituting a block to polyspermy. In addition, there is a block at the level of the oolemma (also known as the membrane block to polyspermy).

Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm.

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.

ADAM2 interactions with mouse eggs and cell lines expressing α4/α9 (ITGA4/ITGA9) integrins: implications for integrin-based adhesion and fertilization.

BACKGROUND: Integrins are heterodimeric cell adhesion molecules, with 18 α (ITGA) and eight ß (ITGB) subunits forming 24 heterodimers classified into five families. Certain integrins, especially the α(4)/α(9) (ITGA4/ITGA9) family, interact with members of the ADAM (a disintegrin and metalloprotease) family. ADAM2 is among the better characterized and also of interest because of its role in sperm function. Having shown that ITGA9 on mouse eggs participates in mouse sperm-egg interactions, we sought to characterize ITGA4/ITGA9-ADAM2 interactions. METHODOLOGY/PRINCIPAL FINDINGS: An anti-ß(1)/ITGB1 function-blocking antibody that reduces sperm-egg binding significantly inhibited ADAM2 binding to mouse eggs. Analysis of integrin subunit expression indicates that mouse eggs could express at least ten different integrins, five in the RGD-binding family, two in the laminin-binding family, two in the collagen-binding family, and ITGA9-ITGB1. Adhesion assays to characterize ADAM2 interactions with ITGA4/ITGA9 family members produced the surprising result that RPMI 8866 cell adhesion to ADAM2 was inhibited by an anti-ITGA9 antibody, noteworthy because ITGA9 has only been reported to dimerize with ITGB1, and RPMI 8866 cells lack detectable ITGB1. Antibody and siRNA studies demonstrate that ITGB7 is the ß subunit contributing to RPMI 8866 adhesion to ADAM2. CONCLUSIONS/SIGNIFICANCE: These data indicate that a novel integrin α-ß combination, ITGA9-ITGB7 (α(9)ß(7)), in RPMI 8866 cells functions as a binding partner for ADAM2. ITGA9 had previously only been reported to dimerize with ITGB1. Although ITGA9-ITGB7 is unlikely to be a widely expressed integrin and appears to be the result of "compensatory dimerization" occurring in the context of little/no ITGB1 expression, the data indicate that ITGA9-ITGB7 functions as an ADAM binding partner in certain cellular contexts, with implications for mammalian fertilization and integrin function.

Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins.

Cell division is inherently mechanical, with cell mechanics being a critical determinant governing the cell shape changes that accompany progression through the cell cycle. The mechanical properties of symmetrically dividing mitotic cells have been well characterized, whereas the contribution of cellular mechanics to the strikingly asymmetric divisions of female meiosis is very poorly understood. Progression of the mammalian oocyte through meiosis involves remodeling of the cortex and proper orientation of the meiotic spindle, and thus we hypothesized that cortical tension and stiffness would change through meiotic maturation and fertilization to facilitate and/or direct cellular remodeling. This work shows that tension in mouse oocytes drops about sixfold during meiotic maturation from prophase I to metaphase II and then increases ∼1.6-fold upon fertilization. The metaphase II egg is polarized, with tension differing ∼2.5-fold between the cortex over the meiotic spindle and the opposite cortex, suggesting that meiotic maturation is accompanied by assembly of a cortical domain with stiffer mechanics as part of the process to achieve asymmetric cytokinesis. We further demonstrate that actin, myosin-II, and the ERM (Ezrin/Radixin/Moesin) family of proteins are enriched in complementary cortical domains and mediate cellular mechanics in mammalian eggs. Manipulation of actin, myosin-II, and ERM function alters tension levels and also is associated with dramatic spindle abnormalities with completion of meiosis II after fertilization. Thus, myosin-II and ERM proteins modulate mechanical properties in oocytes, contributing to cell polarity and to completion of meiosis.

Multivariate analysis of male reproductive function in Inpp5b-/- mice reveals heterogeneity in defects in fertility, sperm-egg membrane interaction and proteolytic cleavage of sperm ADAMs.

Past work indicated that sperm from mice deficient in the inositol polyphosphate 5-phosphatase Inpp5b have reduced ability to fertilize eggs in vitro and reduced epididymal proteolytic processing of the sperm protein A Disintegrin and A Metalloprotease 2 (ADAM2). On the basis of these data, our central working hypothesis was that reduced ADAM cleavage would correlate with reduced sperm-egg binding and fusion and in turn with reduced male fertility in Inpp5b(-/-) mice. Multiple endpoints of reproductive functions [mating trials, in vitro fertilization (IVF) assays and ADAM2 and ADAM3 cleavage] were investigated on a male-by-male basis, with pair-wise correlation analysis used to assess the relationships between these various parameters. Motile sperm from Inpp5b(-/-) mice showed significantly reduced fertilization of zona pellucida-free eggs due to reduced binding to the egg plasma membrane and subsequent fusion. Localization of a mouse sperm protein required for gamete fusion, IZUMO1, appears normal in Inpp5b-null sperm. To our surprise and differing from previous reports, we found that ADAM cleavage was only modestly impaired in numerous Inpp5b-null males and varied between individual animals. Performance in mating trials also differed from past reports. The pair-wise correlation analysis revealed that ADAM2 and ADAM3 cleavage was positively correlated, suggesting that processing of these proteins occurs by related/identical mechanisms, but otherwise, there were few correlations between the reproductive endpoints examined here. Nevertheless, this work provides detailed analysis of the Inpp5b(-/-) phenotype and also a blueprint for multivariate analysis to examine relationships between molecular characteristics and in vitro and in vivo physiological functions.

Egg integrins: back in the game of mammalian fertilization.

Recent data provide insights into the function of egg integrins in mammalian fertilization and address some of the controversies regarding the involvement of these molecules in sperm-egg interaction.