Nonsense-mediated mRNA decay, or NMD, is a quality control mechanism that identifies cytoplasmic mRNAs containing translational termination (stop) codons in specific contexts—either premature termination codons or unusually long 3′ untranslated regions (UTRs)—and targets them for degradation. In recent studies, researchers in different labs have knocked out important genes involved in NMD, the up-frameshift genes Upf2 and Upf3a, and one component of chromatoid bodies, the Tudor domain-containing protein Tdrd6, and examined the consequences for spermatogenesis. Disruption of Upf2 during early stages of spermatogenesis resulted in disappearance of nearly all spermatogenic cells through loss of NMD. However, disruption of Upf2 during postmeiotic stages resulted in decreased long 3′ UTR-mediated NMD but no interruption of exon junction-associated NMD. This difference in NMD targeting is possibly due to increased expression of Upf3a in postmeiotic germ cells that antagonizes the functions of Upf3b and somehow favors long 3′ UTR-mediated NMD. Tying these all together, loss of Tdrd6, a structural component of the germ cell-specific cytoplasmic structures called chromatoid bodies, also resulted in loss of long 3′ UTR-mediated NMD by interfering with UPF1/UPF2 interactions, delocalizing UPF1, and destroying chromatoid body integrity. These results suggest that chromatoid bodies play a specialized role in modulating the NMD machinery in postmeiotic spermatids.

Summary Sentence

Nonsense-mediated decay is how cells eliminate mRNAs having premature stop codons or long 3′ untranslated regions. Recent findings in male germ cells uncover new mechanisms for nonsensemediated decay that shine light on existing models.

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of Gata6 cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6 cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst.

Summary Sentence

The chemokine, CCL24, is involved in spatial arrangement of hypoblast cells in the bovine embryo.

Intrauterine growth retardation (IUGR) is closely related to the later development of type 2 diabetes in adulthood. Excessive activation of N-methly-D-aspartate receptors (NMDARs) causes excitatory neurotoxicity, resulting in neuronal injury or death. Inhibition of NMDARs enhances the glucose-stimulated insulin secretion and survival of islet cells in type 2 diabetic mouse and human islets. Here, we examined whether antenatal blockade of NMDARs by Memantine could decrease the risk of diabetes induced by a high-fat (HF) diet at adulthood in IUGR rats. Pregnant SD rats were assigned to four groups: control, IUGR, Memantine, and Memantine IUGR. The pregnant rats were exposed to hypoxic conditions (FiO₂ = 0.105) for 8 h/day (IUGR group) or given a daily Memantine injection (5 mg/kg, i.p.) before hypoxia exposure from embryonic day (E) 14.5 to E 20.5 (Memantine IUGR). The offspring were fed an HF diet with 60% of the calories from age 4 to 12 weeks. We found that NMDAR mRNAs were expressed in the fetal rat pancreas. An HF diet resulted in a high rate of diabetes at adulthood in the IUGR group. Antenatal Memantine treatment decreased the risk of diabetes at adulthood of rats with IUGR, which was associated with rescued glucose tolerance, increased insulin release, improved the insulin sensitivity, and increased expression of genes related to beta-cell function in the pancreas. Together, our results suggest that antenatal blockade of NMDARs by Memantine in pregnant rats improves fetal development and reduces the susceptibility to diabetes at adulthood in offspring.

Summary Sentence

The excessive activation of NMDARs is involved in the restriction of fetal pancreas development induced by intrauterine hypoxia, and antenatal Memantine treatment attenuates the susceptibility to diabetes induced by an HF diet in IUGR rats.

Three-dimensional (3D) in vitro models have been established to study the physiology and pathophysiology of the endometrium.With emerging evidence that the native extracellular matrix (ECM) provides appropriate cues and growth factors essential for tissue homeostasis,we describe, a novel 3D endometrium in vitro model developed from decellularized human endometrial tissue repopulated with primary endometrial cells. Analysis of the decellularized endometrium using mass spectrometry revealed an enrichment of cell adhesion molecules, cytoskeletal proteins, and ECM proteins such as collagen IV and laminin. Primary endometrial cells within the recellularized scaffolds proliferated and remained viable for an extended period of time in vitro. In order to evaluate the hormonal response of cells within the scaffolds, the recellularized scaffolds were treated with a modified 28-day hormone regimen to mimic the human menstrual cycle. At the end of 28 days, the cells within the endometrial scaffold expressed both estrogen and progesterone receptors. In addition, decidualization markers, IGFBP-1 and prolactin, were secreted upon addition of dibutyryl cyclic AMP indicative of a decidualization response. This 3D model of the endometrium provides a new experimental tool to study endometrial biology and drug testing.

Summary Sentence

Primary endometrial cells within a recellularized scaffold respond to a 28-day menstrual cycle.

Metabolic rich and poor conditions are both characterized by elevated free fatty acid levels and have been associated with impaired female fertility. In particular, saturated free fatty acids have a dose-dependent negative impact on oocyte developmental competence, while monounsaturated free fatty acids appear less harmful. Cumulus cells seem to protect the oocyte against free fatty acids, and the aim of this study was to determine the mechanism behind this protection In particular, the role of the enzyme stearoyl-CoA desaturase (SCD) that converts saturated into monounsaturated fatty acids was investigated. SCD gene and protein were abundantly expressed in cumulus cells, but expression was low in oocytes. The level of SCD protein expression in cumulus cells did not change when COCs were exposed to saturated stearic acid during maturation. SCD inhibition in the presence of stearic acid significantly reduced the developmental competence of oocytes and increased the incidence of apoptosis in cumulus cells. The esterified oleic/stearic acid ratio of the neutral lipid fraction in cumulus cells decreased in the presence of SCD inhibitors when COCs were exposed to saturated free fatty acids during maturation, indicating the SCD-specific conversion of saturated fatty acids under noninhibiting conditions. The observation that cumulus cells can desaturate the potentially toxic stearic acid into oleic acid via SCD activity provides a mechanistic insight into how the cumulus cells protect the oocyte against toxicity by saturated fatty acid.

Summary Sentence

Stearoyl-CoA desaturase in bovine cumulus cells converts saturated into monounsaturated fatty acid and protects the oocyte against fatty acid-induced lipotoxicity.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the “9 2” microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice.

The luteinizing hormone receptor (LHCGR) is necessary for fertility, and genetic mutations cause defects in reproductive development and function. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP). We have previously characterized a mouse model (KiLHR^D582G) for FMPP that exhibits the same phenotype of precocious puberty, Leydig cell hyperplasia, and elevated testosterone as boys with the disorder. We observed that KiLHR^D582G male mice became infertile by 6 months of age, although sperm count and motility were normal. In this study, we sought to determine the reason for the progressive infertility and the long-term consequences of constant LHCGR signaling. Mating with superovulated females showed that infertile KiLHR^D582G mice had functional sperm and normal accessory gland function. Sexual behavior studies revealed that KiLHR^D582G mice mounted females, but intromission was brief and ejaculation was not achieved. Histological analysis of the reproductive tract showed unique metaplastic changes resulting in pseudostratified columnar epithelial cells with cilia in the ampulla and chondrocytes in the penile body of the KiLHR^D582G mice. The infertile KiLHR^D582G exhibited enlarged sinusoids and a decrease in smooth muscle content in the corpora cavernosa of the penile body. However, collagen content was unchanged. Leydig cell adenomas and degenerating seminiferous tubules were seen in 1-year-old KiLHR^D582G mice. We conclude that progressive infertility in KiLHR^D582G mice is due to sexual dysfunction likely due to functional defects in the penis.

Summary Sentence

Activating mutation in LHCGR causes age-related sexual dysfunction and testicular tumors.

Although in vitro exposure to physiological concentrations of glucorticoids did not affect maturation of mouse oocytes, it significantly inhibited nuclear maturation of pig oocytes. Studies on this species difference in oocyte sensitivity to glucocorticoids will contribute to our understanding of how stress/glucocorticoids affect oocytes. We showed that glucorticoid receptors (NR3C1) were expressed in both oocytes and cumulus cells (CCs) of both pigs and mice; however, while cortisol inhibition of oocyte maturation was overcome by NR3C1 inhibitor RU486 in pigs, it could not be relieved by RU486 in mice. The mRNA level of 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) was significantly higher than that of HSD11B2 in pig cumulus-oocyte complexes (COCs), whereas HSD11B2 was exclusively expressed in mouse COCs. Pig and mouse cumulus-denuded oocytes (DOs) expressed HSD11B2 predominantly and exclusively, respectively. In the presence of cortisol, although inhibiting HSD11B2 decreased maturation rates of COCs in both species, inhibiting HSD11B1 improved maturation of pig COCs while having no effect on mouse COCs. Cortisolcortisone interconversion observation confirmed high HSD11B1 activities in pig oocytes but none in mouse oocytes, a higher HSD11B2 activity in mouse than in pig oocytes, and a rapid cortisolcortisone interconversion in pig COCs catalyzed by HSD11B1 from CCs and HSD11B2 from DOs. In conclusion, the species difference in glucocorticoid sensitivity between pig and mouse oocytes is caused by their different contents/ratios of HSD11B1 and HSD11B2, which maintain different concentrations of active glucocorticoids. While cortisol inhibited pig oocytes by interacting with NR3C1, glucocorticoid suppression of mouse oocytes was apparently not mediated by NR3C1.

Summary Sentence

The species difference in glucocorticoid sensitivity between pig and mouse oocytes is caused by their different contents/ratios of HSD11B1 and 2, and glucocorticoids impair maturation of mouse oocytes apparently not mediated by NR3C1.

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

Summary Sentence

Lpxrfa regulates reproduction in the zebrafish brain-pituitary axis through inhibitory effects on gonadotropins and Gnrh3, and Lpxrfa elicits these effects by utilizing Lpxrf receptors, as well as receptors of other reproductive neuropeptides.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle—stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Summary Sentence

KP10 increased all three gonadotropin subunit promoters with an increasing expression of ADCYAP1 type I receptor in pituitary gonadotroph LbetaT2 cells overexpressing kisspeptin receptor.

The phosphoinositide 3-kinase/AKT (protein kinase B) signaling pathway negatively regulates follicle activation via the forkhead box O (FOXO) transcription factor in rodents. FOXO3 knockout mice exhibit global activation of primordial follicles leading to early depletion of ovarian follicles and subsequent infertility. Whether a similar mechanism for follicle activation exists in the primate ovary is unclear. In the current study, protein localization of FOXO1, 3, and 4 as well as their upstream regulator, AKT/p-AKT, was examined in rhesus macaque ovaries of three developmental stages: fetal, prepubertal, and adult. FOXO1 protein is expressed in granulosa cells of fetal, prepubertal, and adult ovaries. FOXO3 is distributed sparsely in the mitotically active germ cells, but its expression decreases following follicle formation in the macaque fetal ovary. In addition, FOXO3 is seldom with interanimal variation in the prepubertal ovary and is absent in the adult ovary. FOXO4 is nondetectable in fetal ovaries, although it is expressed in some theca cells of antral follicles and some stromal cells in prepubertal and adult ovaries. Our results suggest that the regulation and/or function of FOXO3 in the primate primordial follicle may differ than that of the rodent. Nevertheless, AKT/p-AKT is expressed in macaque primordial oocytes, suggesting that similar upstream events but different downstream effects may regulate primordial follicle activation in nonhuman primates compared to rodents. Elucidation of the mechanism responsible for follicle activation in primates will be crucial for understanding primary ovarian insufficiency, improving female fertility, and applying techniques for in vitro maturation of follicles for fertility preservation in cancer survivors.

Summary Sentence

FOXO3 protein localizes in the oocyte of fetal and prepubertal but not adult ovaries while FOXO1 and 4 are expressed in granulosa and theca cells, receptively in the rhesus macaque.

The observation of pups born from recipient and donor mice after ovariectomy followed by ovarian transplant poses the interesting possibility of an extraovarian source of oocytes. However, whether mammalian adult oocytes reside in extragonadal tissues remains elusive. Using transgenic fluorescent reporter mice and transplantation surgeries, we demonstrate the presence of both donorand recipient-derived corpora lutea and recovery of both donor- and recipient-derived offspring from ovariectomized mice after transplantation of donor ovaries. A potential region for extraovarian oocytes is the hilum, a ligament-like structure between the ovary and the reproductive tract. Immunofluorescent confocal microscopy of mouse ovaries and reproductive tracts revealed that a population of primordial follicles resides outside the ovary within the hilum. Ovariectomy-only controls confirmed that oocytes remain in the recipient hilum after surgery. These results provide evidence that the hilum is a reserve source of follicles, which likely return to the ovary for maturation and ovulation. By identifying a new follicle reservoir, our study addresses a long-standing question in reproductive biology and contributes to new conceptual knowledge about ovarian function and fertility.

Summary Sentence

An extraovarian source of oocytes exists within the mouse ovarian hilum, amuscular structure that connects the ovary to the reproductive tract; these oocytes reside within primordial and primary follicles and may contribute to female fertility.

We investigated the interaction between prenatal nicotine exposure and intrauterine infection using established rat models. Beginning at gestation day (GD) 6, dams were continuously infused with either saline or 6 mg/kg/day nicotine (Nic). At GD 14, dams received either sterile broth or 105 colony-forming units Mycoplasma pulmonis (MP), resulting in four treatment groups: control (4 dams, 33 fetal units); MP only (5 dams, 55 fetal units); Nic only (5 dams, 61 fetal units), and Nic MP (7 dams, 82 fetal units). AtGD18, nicotine exposure significantly increased (P≤0.02) the percentage of amniotic fluids and fetuses infected by MP but did not impact colonization rates of maternal sites. Nicotine exposure significantly reduced the numbers of MP in the placenta required for high microbial loads (≥10⁴ color-changing units) in the amniotic fluid (P < 0.01). Fetal inflammatory response lesions were most extensive in the Nic only and Nic MP groups (P < 0.0001). Control and MP only placentas were interleukin (IL)10-dominant, consistent with an M2/Th2 environment. Placentas exposed to nicotine shifted to a neutral environment, with equivalent levels of interferon gamma (IFNG) and IL10. Both IL6 and tumor necrosis factor (TNF) levels in amniotic fluid were highly elevated when both nicotine and infection were present. Our study suggests that prenatal exposure to nicotine increases the risk for intrauterine infection, lowers the infectious dose required to breach the placental barrier and infect the amniotic fluid and fetus, and alters the pathology and inflammatory profile associated with maternal and fetal sites.

Summary Sentence

A modifiable risk factor (nicotine) can directly impact what has been considered an immutable risk factor (infection) resulting in increased risk of pathogen transmission across the placental barrier with subsequent impacts on placental pathology and maternal/fetal immune environment.

Overnutrition during pregnancy could increase risks of cardiovascular diseases in late life. This study investigated whether and how reactive oxygen species (ROS) may influence functions of large-conductance Ca² -activated K channels (BK_Ca) in the offspring exposed to prenatal high sucrose (HS). We found that prenatal HS diets significantly increased phenylephrine (PE)-induced vessel contractions in mesenteric arteries of the adult offspring. Pretreatment with iberiotoxin (BK_Ca blocker, IBTX) significantly increased PE-mediated vascular contractions in the control, not in the HS group. Electrophysiological studies demonstrated that BK_Ca current density and singlechannel current were reduced in the vascular smooth muscle cells (VSMCs) of the HS offspring. The expression of BK_Ca alpha, beta1 subunits in mesenteric arteries was decreased in the HS offspring, indicating that both activity and number of BK_Ca channels in HS offspring were reduced. Superoxide production and NADPH oxidase (NOX)4 of the HS offspring were elevated. Following inhibiting NOX by apocynin, vasoconstriction in the HS offspring was weakened and the reduced currents in the VSMCs were improved with altered protein kinase B (AKT) pathway. The results suggested that NOX4-derived ROS might inhibit the offspring vascular BK_Ca channel activity via AKT pathway.

Summary Sentence

Large-conductance Ca² -activated K channels of mesenteric arteries in the offspring exposing to maternal high-sucrose intake were impaired, which might be involved in altered NOX4-derived ROS and AKT/FBXO32 pathway.

Meiotic recombination ensures faithful segregation of homologous chromosomes during meiosis and generates genetic diversity in gametes. MEIOB (meiosis specific with OB domains), a meiosis specific single-stranded DNA-binding homolog of replication protein A1 (RPA1), is essential for meiotic recombination. Here, we investigated the molecular mechanisms of MEIOB by characterizing its binding partners spermatogenesis associated 22 (SPATA22) and RPA. We find that MEIOB and SPATA22 form an obligate complex and contain defined interaction domains. The interaction between these two proteins is unusual in that nearly any deletion in the binding domains abolishes the interaction. Strikingly, a single residue D383 in MEIOB is indispensable for the interaction. The MEIOB/SPATA22 complex interacts with the RPA heterotrimeric complex in a collaborative manner. Furthermore, MEIOB and SPATA22 are recruited to induced DNA double-strand breaks (DSBs) together but not alone. These results demonstrate the cooperative property of the MEIOB-SPATA22 complex in its interaction with RPA and recruitment to DSBs.

Summary Sentence

MEIOB and SPATA22 function as a novel meiosis-specific heterocomplex.

Dibutyl phthalate (DBP) is present in consumer products and the coating of some oralmedications. Acetyl tributyl citrate (ATBC) has been proposed as an alternative to DBP because DBP causes endocrine disruption in animal models. Following ingestion, DBP is converted to its main metabolite mono-butyl phthalate (MBP) which has been detected in >90% of human follicular fluid samples. Previous studies show that DBP reduces the number of antral follicles present in the ovaries of mice. Thus, this study was designed to evaluate the effects of DBP, MBP, and ATBC on in vitro growth and viability of mouse ovarian antral follicles. Antral follicles were isolated from CD-1 females (PND32-37) and treated with vehicle, DBP, MBP, or ATBC (starting at 0.001 and up to 1000µg/ml for DBP; 24–72 h). Follicle diameter, ATP production, qPCR, and TUNEL were used to measure follicle growth, viability, cell cycle and apoptosis gene expression, and cell death-associated DNA fragmentation, respectively. While MBP did not cause toxicity, DBP exposure at ≥10µg/ml resulted in growth inhibition followed by cytoxicity at ≥500 µg/ml. ATBC increased the number of nongrowing follicles at 0.01 µg/ml and did not affect ATP production, but increased TUNEL positive area in treated follicles. Gene expression results suggest that cytotoxicity in DBP-treated follicles occurs via activation of cell cycle arrest prior to follicular death. These findings suggest that concentrations of DBP ≥10 µg/ml are detrimental to antral follicles and that ATBC should be examined further as it may disrupt antral follicle function at low concentrations.

Summary Sentence

DBP and ATBC exposures resulted in dose-specific follicle growth inhibition with high concentrations of DBP causing cytoxicity via activation of cyclin-dependent kinase inhibitors and subsequent apoptosis.

The legend for Figure 7 was incorrectly placed under Figure 8. The legend for Figure 8 was incorrectly placed under Figure 7. This has now been corrected and the correct legends are under Figures 7 and 8. The author regrets the error. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com