Genome analyses of single human oocytes.

Single-cell genome analyses of human oocytes are important for meiosis research and preimplantation genomic screening. However, the nonuniformity of single-cell whole-genome amplification hindered its use. Here, we demonstrate genome analyses of single human oocytes using multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technology. By sequencing the triads of the first and second polar bodies (PB1 and PB2) and the oocyte pronuclei from same female egg donors, we phase the genomes of these donors with detected SNPs and determine the crossover maps of their oocytes. Our data exhibit an expected crossover interference and indicate a weak chromatid interference. Further, the genome of the oocyte pronucleus, including information regarding aneuploidy and SNPs in disease-associated alleles, can be accurately deduced from the genomes of PB1 and PB2. The MALBAC-based preimplantation genomic screening in in vitro fertilization (IVF) enables accurate and cost-effective selection of normal fertilized eggs for embryo transfer.

Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

Cell Repolarization: A Bifurcation Study of Spatio-Temporal Perturbations of Polar Cells.

The intrinsic polarity of migrating cells is regulated by spatial distributions of protein activity. Those proteins (Rho-family GTPases, such as Rac and Rho) redistribute in response to stimuli, determining the cell front and back. Reaction-diffusion equations with mass conservation and positive feedback have been used to explain initial polarization of a cell. However, the sensitivity of a polar cell to a reversal stimulus has not yet been fully understood. We carry out a PDE bifurcation analysis of two polarity models to investigate routes to repolarization: (1) a single-GTPase ("wave-pinning") model and (2) a mutually antagonistic Rac-Rho model. We find distinct routes to reversal in (1) vs. (2). We show numerical simulations of full PDE solutions for the RD equations, demonstrating agreement with predictions of the bifurcation results. Finally, we show that simulations of the polarity models in deforming 1D model cells are consistent with biological experiments.

DNA methylation analysis and editing in single mammalian oocytes.

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy )-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy -related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.

FASCIN regulates actin assembly for spindle movement and polar body extrusion in mouse oocyte meiosis.

During mouse oocyte meiotic maturation, actin filaments play multiple roles in meiosis such as spindle migration and cytokinesis. FASCIN is shown to be an actin-binding and bundling protein, making actin filaments tightly packed and parallel-aligned, and FASCIN is involved in several cellular processes like adhesion and migration. FASCIN is also a potential prognostic biomarker and therapeutic target for the treatment of metastatic disease. However, little is known about the functions of FASCIN in oocyte meiosis. In the present study, we knocked down the expression of FASCIN, and our results showed that FASCIN was essential for oocyte maturation. FASCIN was all expressed in the different stages of oocyte meiosis, and it mainly localized at the cortex of oocytes from the GV stage to the MII stage and showed a similar localization pattern with actin and DAAM1. Depletion of FASCIN affected the extrusion of the first polar body, and we also observed that some oocytes extruded from the large polar bodies. This might have resulted from the defects of actin assembly, which further affected the meiotic spindle positioning. In addition, we showed that inhibition of PKC activity decreased FASCIN expression, indicating that FASCIN might be regulated by PKC. Taken together, our results provided evidence for the important role of FASCIN on actin filaments for spindle migration and polar body extrusion in mouse oocyte meiosis.

Assisted oocyte activation effects on the morphokinetic pattern of derived embryos.

OBJECTIVE: Assisted oocyte activation (AOA) can restore fertilization rates after IVF/ICSI cycles with fertilization failure. AOA is an experimental technique, and its downstream effects remain poorly characterized. Clarifying the relationship between AOA and embryo, morphokinetics could offer complementary insights into the quality and viability of the embryos obtained with this technique. The aim of this study is to compare the preimplantation morphokinetic development of embryos derived from ICSI-AOA (experimental group) vs. ICSI cycles (control group). METHODS: A retrospective cohort study was carried out with 141 embryos from fresh oocyte donation cycles performed between 2013 and 2017; 41 embryos were derived from 7 ICSI-AOA cycles and 100 embryos from 18 ICSI cycles. Morphokinetic development of all embryos was followed using a time-lapse system. RESULTS: We show that embryos from both groups develop similarly for most milestones, with the exception of the time of second polar body extrusion (tPB2) and the time to second cell division (t3). CONCLUSIONS: We conclude that ionomycin mediated AOA does not seem to affect the morphokinetic pattern of preimplantation embryo development, despite the alterations found in tPB2 and t3, which could directly reflect the use of a Ca2+ ionophore as a transient and quick non-physiologic increase of free intracytoplasmic Ca2+.

Early rescue oocyte activation for activation-impaired oocytes with no second polar body extrusion after intracytoplasmic sperm injection.

PURPOSE: When rescue artificial oocyte activation (ROA) is performed on the day after intracytoplasmic sperm injection (ICSI) or later, embryonic development is poor and seldom results in live births. The efficacy of an early ROA after ICSI is unclear. Is early ROA effective in rescuing unfertilized oocytes that have not undergone second polar body extrusion several hours after ICSI? METHODS: We performed retrospective cohort study between October 2016 and September 2019, targeting 2891 oocytes in 843 cycles when ICSI was performed. We performed ROA with calcium ionophore on 395 of the 475 oocytes with no second polar extrusion 2.5-6 h after ICSI. RESULTS: The normal fertilization rate of ROA oocytes was significantly higher than non-ROA oocytes (65.8% vs 6.7%, P < 0.001). The blastocyst development rate in ROA oocytes was significantly lower than spontaneously activated oocytes (48.9% vs 67.2%, P < 0.001). The ROA oocyte implantation rate did not significantly differ from the spontaneously activated oocytes (36.0% vs 41.2%). We observed no differences in the implantation rates and blastocyst development rates over the 2.5-6 h from ICSI until ROA. CONCLUSION: Early ROA is effective, and the optimal timing appears to be 2.5-6 h after ICSI.

Ribonucleic Acid Export 1 Is a Kinetochore-Associated Protein That Participates in Chromosome Alignment in Mouse Oocytes.

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore-microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.

Phospholipase C inhibits apoptosis of porcine oocytes cultured in vitro.

Oocyte apoptosis can be used as an indicator of oocyte quality and development competency. Phospholipase C (PLC) is a critical enzyme that participates in phosphoinositide metabolic regulation and performs many functions, including the regulation of reproduction. In this study, we aimed to explore whether PLC participates in the regulation of apoptosis in porcine oocytes and investigated its possible mechanism. In porcine oocytes, 0.5 µM U73122 (the PLC inhibitor) was considered to be the best concentration to facilitate maturation, and 0.5 µM m-3M3FBS (the PLC activator) was regarded as the most appropriate concentration to inhibit maturation. The percentage of cleavage and blastocysts treated with 0.5 µM U73122 was lower than that of the control group. Furthermore, the percentage of cleavage and blastocysts treated with 0.5 µM m-3M3FBS was higher than that of the control group. The relative PLC messenger RNA (mRNA) expression tested by a quantitative real-time polymerase chain reaction was found to be inhibited by 0.5 µM U73122 or activated by 0.5 µM m-3M3FBS. The relative mRNA abundance of BAK, BAX, CASP3, CASP8, and TP53 and protein abundance of Bak, cleaved caspase-3, caspase-8, and P53 was activated by U73122 or inhibited by m-3M3FBS, while the relative mRNA and protein level of BCL6 showed the opposite trend. The intracellular Ca2+ concentration increased and the expression of PLCB1 protein also increased in porcine oocytes when they were cultured with 0.5 µM m-3M3FBS for 44 hours. The abundance of proteins PKCß and CAMKIIα and the expression of several downstream genes (CDC42, NFATc1, NFATc2, NFκB, and NLK) were activated by m-3M3FBS or inhibited by U73122. Our findings indicate that PLC inhibits apoptosis and maturation in porcine oocytes. The intracellular Ca2+ concentration, two Ca2+ -sensitive proteins, and several downstream genes were positively regulated by PLC.

Morphokinetics and in vitro developmental potential of monopronucleated ICSI zygotes until the blastocyst stage.

The aim of this study was to provide a more comprehensive understanding of 1PN intracytoplasmic sperm injection (ICSI) zygotes. To achieve this objective, we assessed whether all 1PN-derived embryos showed a similar morphokinetic pattern, and if the morphokinetic behaviour of 1PN-derived embryos was comparable with that of 2PN-derived embryos. In total, 149 1PN ICSI zygotes (study group) and 195 2PN ICSI zygotes (control group) were included in the study. Embryo development potential was evaluated in terms of blastocyst rate. Morphokinetic parameters, including the pronucleus diameter and kinetics of in vitro development, were also analyzed. Embryos derived from 1PN ICSI zygotes showed impaired development compared with 2PN-derived embryos, with blastocyst rates of 28.9% and 67.2%, respectively. The diameter of the pronucleus of 1PN zygotes was larger than that of 2PN zygotes. When compared with 2PN-derived embryos, those derived from 1PN zygotes had a visible pronucleus for a shorter time, in addition to a longer syngamy time and slower kinetic behaviour from two to nine cells. When 1PN-derived blastocysts and 2PN-derived blastocysts were compared, the developmental kinetics were similar in both groups, except for a delayed and longer duration of the compaction phase in 1PN-derived embryos. In conclusion, monopronucleated ICSI zygotes present differences in developmental capacity and morphokinetic behaviour compared with 2PN ICSI zygotes, showing particular morphokinetic parameters related to pronucleus formation. Only the 1PN ICSI-derived embryos that reached the blastocyst stage have similar morphokinetic development to blastocysts from 2PN zygotes.

Vesicular transport protein Arf6 modulates cytoskeleton dynamics for polar body extrusion in mouse oocyte meiosis.

Arf6 (ADP-ribosylation factor 6) is known to play important roles in membrane dynamics through the regulation of actin filament reorganization for multiple cellular processes such as cytokinesis, phagocytosis, cell migration and tumor cell invasion. However, the functions of Arf6 in mammalian oocyte meiosis have not been clarified. In present study we showed that Arf6 expressed in mouse oocytes and was mainly distributed around the spindle during meiosis. Depletion of Arf6 by morpholino microinjection caused oocytes failing to extrude first polar body. Further analysis indicated that Arf6 knock down caused the aberrant actin distribution, which further induced the failure of meiotic spindle movement. And the loss of oocyte polarity also confirmed this. The regulation of Arf6 on actin filaments in mouse oocytes might be due to its effects on the phosphorylation level of cofilin and the expression of Arp2/3 complex. Moreover, we found that the decrease of Arf6 caused the disruption of spindle formation, indicating the multiple roles of Arf6 on cytoskeleton dynamics in meiosis. In summary, our results indicated that Arf6 was involved in mouse oocyte meiosis through its functional roles in actin-mediated spindle movement and spindle organization.

Loss of PKC mu function induces cytoskeletal defects in mouse oocyte meiosis.

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6-month-old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.

Tributyltin oxide exposure impairs mouse oocyte maturation and its possible mechanisms.

Tributyltin oxide (TBTO) has been widely used as marine antifouling composition, preservative, biocide, and a stabilizer in plastic industry. Previous studies have indicated that TBTO can cause immunotoxicity as an environmental pollutant. However, little is known about its reproductive toxicity, especially on female oocyte maturation and the underlying mechanisms. In this study, mouse oocytes were cultured with different concentrations of TBTO in vitro, and several crucial events during meiotic maturation were evaluated. We found that the first polar body extrusion rate was significantly reduced, which reflected the disruption of meiotic maturation. The rate of abnormal spindle organization increased significantly, accompanied with a higher rate of chromosome misalignment. In addition, TBTO treatment increased reactive oxygen species generation markedly, which also accelerated the early-stage apoptosis. Moreover, heterogeneous mitochondrial distribution, mitochondrial dysfunction, and higher rate of aneuploidy were detected, which consequently disrupted in vitro fertilization. In conclusion, our results indicated that TBTO exposure could impair mouse oocyte maturation by affecting spindle organization, chromosome alignment, mitochondria functions, oxidative stress, and apoptosis.

Allicin protects porcine oocytes against damage during aging in vitro.

Allicin, a chemical component of garlic, has strong antioxidant activity and is thought to exert antiaging effects in vitro. We investigated whether allicin treatment would protect porcine oocytes and embryos from postovulatory aging mediated by apoptosis and autophagy. The rates of oocyte survival and polar body extrusion in samples treated with 1 µM allicin (1 AL) were significantly higher than in untreated samples (0 AL). In addition, 1 AL prevented defects in spindle formation and chromosome alignment, as well as decreases in the expression of maturation markers, during in vitro aging. In this study, we considered allicin to be a regulator of autophagy rather than an antioxidant or antiapoptotic agent. At the embryo level, although the cleavage rate after parthenogenetic activation was similar in all groups, the blastocyst formation rate was higher in the 1 AL group than in the 0 AL group. Our findings demonstrate that allicin effectively prevents the deterioration of porcine oocytes during aging in vitro, and could therefore be used to improve the quality of aged oocytes used in in vitro experiments.

The small GTPase RhoA regulates the LIMK1/2-cofilin pathway to modulate cytoskeletal dynamics in oocyte meiosis.

LIM kinases (LIMK1/2) are LIM domain-containing serine/threonine/tyrosine kinases that mediate multiple cellular processes in mitosis. In the present study, we explored the functional roles and potential signaling pathway of LIMK1/2 during mouse oocyte meiosis. Disruption of LIMK1/2 activity and expression significantly decreased oocyte polar body extrusion. Live-cell imaging revealed that spindle migration was disturbed after both LIMK1 and LIMK2 knock down, and this might be due to aberrant distribution of actin filaments in the oocyte cytoplasm and cortex. Meanwhile, our results demonstrated that the function of LIMK1 and LIMK2 in actin assembly was related to cofilin phosphorylation levels. In addition, disruption of LIMK1/2 activity significantly increased the percentage of oocytes with abnormal spindle morphologies, which was confirmed by the abnormal p-MAPK localization. We further, explored the upstream molecules of LIMK1/2, and we found that after depletion of ROCK, phosphorylation of LIMK1/2 and cofilin were significantly decreased. Moreover, RhoA inhibition caused the decreased expression of ROCK, p-LIMK1/2, and cofilin. In summary, our results indicated that the small GTPase RhoA regulated LIMK1/2-cofilin to modulate cytoskeletal dynamics during mouse oocyte meiosis.

Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.

Chromosome segregation in meiosis is controlled by a conserved pathway that culminates in Separase-mediated cleavage of the α-kleisin Rec8, leading to dissolution of cohesin rings. Drosophila has no gene encoding Rec8, and the absence of a known Separase target raises the question of whether Separase and its regulator Securin (Pim in Drosophila) are important in Drosophila meiosis. Here, we investigate the role of Securin, Separase and the cohesin complex in female meiosis using fluorescence in situ hybridization against centromeric and arm-specific sequences to monitor cohesion. We show that Securin destruction and Separase activity are required for timely release of arm cohesion in anaphase I and centromere-proximal cohesion in anaphase II. They are also required for release of arm cohesion on polar body chromosomes. Cohesion on polar body chromosomes depends on the cohesin components SMC3 and the mitotic α-kleisin Rad21 (also called Vtd in Drosophila). We provide cytological evidence that SMC3 is required for arm cohesion in female meiosis, whereas Rad21, in agreement with recent findings, is not. We conclude that in Drosophila meiosis, cohesion is regulated by a conserved Securin-Separase pathway that targets a diverged Separase target, possibly within the cohesin complex.

Profilin 1 plays feedback role in actin-mediated polar body extrusion in mouse oocytes.

Mammalian oocytes undergo several crucial processes during meiosis maturation, including spindle formation and migration and polar body extrusion, which rely on the regulation of actin. As a small actin-binding protein, profilin 1 plays a central role in the regulation of actin assembly. However, the functions of profilin 1 in mammalian oocytes are uncertain. To investigate the function of profilin 1 in oocytes, immunofluorescent staining was first used to examine profilin 1 localisation. The results showed that profilin 1 was localised around the meiotic spindles and was colocalised with cytoplasmic actin. Knockdown (KD) of profilin 1 with specific morpholino microinjection resulted in failure of polar body extrusion. This failure resulted from an increase of actin polymerisation both at membranes and in the cytoplasm. Furthermore, western blot analysis revealed that the expression of Rho-associated kinase (ROCK) and phosphorylation levels of myosin light chain (MLC) were significantly altered after KD of profilin 1. Thus, the results indicate that a feedback mechanism between profilin, actin and ROCK-MLC2 regulates actin assembly during mouse oocyte maturation.

Impact of polar body biopsy on embryo morphokinetics-back to the roots in preimplantation genetic testing?

PURPOSE: Polar body biopsy (PBB) is a common technique in preimplantation genetic testing (PGT) to assess the chromosomal status of the oocyte. Numerous studies have been implemented to investigate the impact of biopsies on embryo development; however, information on embryo morphokinetics is still lacking. Hence, we investigated the impact of PBB on morphokinetic parameters in early embryo development. METHODS: Four hundred four embryos (202 PBB, 202 control) were retrospectively analyzed. Patients were stimulated with a gonadotropin-releasing hormone antagonist ovarian hyperstimulation protocol. After fertilization check, embryos were incubated in a time-lapse incubator. The groups were matched for maternal age at time of oocyte retrieval. RESULTS: Mean group times for reaching specific developmental time points showed no significant difference comparing embryos with PBB conducted and without. Likewise, further subdivision of the PBB group in euploid and aneuploid embryos revealed no differences in the early embryo morphokinetic development compared to the control group. Aneuploidy testing revealed a high prevalence of chromosomal aberrations for chromosomes 21, 4, 16, and 19. CONCLUSIONS: In conclusion, PBB does not impact the morphokinetic parameters of the embryo development. PBB can be safely applied without the risk of impairing the reproductive potential of the embryo and can be highly recommended as safe and practicable PGT approach, especially in countries with prevailing restrictions regarding PGT analysis.

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes.

Higher Order Oligomerization of the Licensing ORC4 Protein Is Required for Polar Body Extrusion in Murine Meiosis.

We have previously shown that the DNA replication licensing factor ORC4 forms a cage around the chromosomes that are extruded in both polar bodies during murine oogenesis, but not around the chromosomes that are retained in the oocyte or around the sperm chromatin. We termed this structure the ORC4 cage. Here, we tested whether the formation of the ORC4 cage is necessary for polar body extrusion (PBE). We first experimentally forced oocytes to extrude sperm chromatin as a pseudo-polar body and found that under these conditions the sperm chromatin did become enclosed in an ORC4 cage. Next, we attempted to prevent the formation of the ORC4 cage by injecting peptides that contained sequences of different domains of the ORC4 protein into metaphase II (MII) oocytes just before the cage normally forms. Our rationale was that the ORC4 peptides would block protein-protein interactions required for cage formation. Two out of six tested peptides prevented the ORC4 cage formation and simultaneously inhibited PBE, resulting in the formation of two pronuclei (2 PN) that were retained in the oocyte. Together, these data demonstrate that ORC4 oligomerization is required to form the ORC4 cage and that it is required for PBE. J. Cell. Biochem. 118: 2941-2949, 2017. © 2017 Wiley Periodicals, Inc.