Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

STUDY QUESTION: Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? SUMMARY ANSWER: Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. WHAT IS KNOWN ALREADY: Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.

STUDY QUESTION: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER: Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY: O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION: This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. LIMITATIONS, REASONS FOR CAUTION: In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS: This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence.

The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus-oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P<0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P<0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Fractures after stroke: frequency, types, and associations.

BACKGROUND AND PURPOSE: Stroke patients may have an increased risk of fractures because of weak bones or an increased risk of falling. Our goal was to estimate the frequency of fracture after stroke and to identify those at greatest risk. METHODS: This study incorporated 2 complementary strategies: a prospective, single-center, cohort study and an analysis of Scottish routine hospital discharge data. RESULTS: Eighty-eight fractures (30% hip) occurred in 2696 hospital-referred stroke patients. The proportions sustaining any fracture or hip fracture within 2 years were 4% and 1.1%, respectively, 1.4 (95% CI, 0.92 to 2.07) times the rate of hip fracture in the general population (ie, observed number divided by expected number or standardized morbidity ratio). Female sex, older age, low abbreviated mental test score, and prestroke dependence were associated with an increased hip fracture rate. Routine data identified 129 935 acute stroke patients admitted to Scottish hospitals. During 363 447 patient-years, 4528 patients had hip fractures, 2.0% had fractures by 1 year, and 10.6% had fractures by 10 years. This is 1.7 times the rate of hip fracture in the general population and 2.3 times that in patients with myocardial infarction. Older patients predictably had the highest rate of poststroke hip fractures but a lower standardized morbidity ratio than younger patients. CONCLUSIONS: Fractures after stroke are probably frequent and serious enough to justify the development of preventive strategies, but the modest event rate would mean that randomized, controlled trials to test these strategies specifically in stroke patients would need to enroll thousands of patients.

Mortality from congenital malformations by mother's country of birth.

Mortality from congenital malformations by mother's country of birth was examined in England and Wales between the years 1976 and 1980, based on stillbirths and infant deaths. There were 18 870 stillbirths and infant deaths attributed to congenital malformations in this period, of which 2 375 (13%) were to mothers born outside the United Kingdom. There were excess deaths from malformations among Pakistani, Indian/Bangladeshi, African, and Irish mothers. In contrast, West Indian mothers had a consistent deficit in deaths from malformations over the study period. The significance of these findings is discussed.

Testicular cancer mortality in England and Wales 1971-80: variations by occupation.

The occupations of all males aged 15 and over dying of cancer of the testis in England and Wales from 1971 to 1980 (2434 cases) have been examined in a case control study. Significant excess mortality was found in farmers (although not farm workers), food manufacture and preparation workers, and draughtsmen, although the results show little evidence of a major role for occupation in the aetiology of this cancer. The results may, however, aid the search for those factors in modern life that may have contributed to the sharp rise in incidence of this cancer in recent years.

Maternal drug histories and congenital malformations: limb reduction defects and oral clefts.

In a case control study, prescription data were examined for the three months before the last menstrual period and for the first trimester of pregnancy in (a) 115 mothers of children with limb reduction defects, (b) 676 mothers of children with oral cleft, and (c) an equal number of control mothers of normal babies from the same doctor's practice for each case. In the limb reduction study, the study mothers were prescribed more drugs generally although this did not reach statistical significance, nor were there significant differences between study and control mothers for individual groups of drugs. In the oral cleft study, significantly more drugs were prescribed to study mothers in the three months before the last menstrual period, and a similar trend, which did not reach statistical significance, was observed in the first trimester. Anticonvulsant drugs were prescribed significantly more frequently to study mothers during the whole period of the study. A significant association was also demonstrated between oral contraceptives taken in the three months before the last menstrual period and oral cleft, but doubt must remain concerning this relationship; the risk is not well understood and is likely to be nonspecific. A number of other significant associations were identified, although their importance in practice is uncertain in view of the confounding factors that may affect a study of this kind.

The effects of short-term manipulation of thyroid hormone status coinciding with primary wool follicle development on fleece characteristics in Merino sheep.

Thyroidectomy surgery performed late in gestation results in perturbations in wool follicle development in foetal sheep, showing the importance of thyroid hormones for wool follicle development. The aim of this study was to determine the influence of transient manipulation of thyroid hormone status at a time corresponding with foetal primary wool follicle initiation. Pregnant Merino ewes (n = 12 per treatment) were treated daily between gestational days 55 and 64 with control (vehicle), exogenous thyroxine (T4) or propylthiouracil (PTU), an inhibitor of T4 synthesis, and conversion to the active form of the thyroid hormone (triiodothyronine). There were no significant differences in birth weight, gestational lengths and birth coat scores of the resultant lambs. The total primary and secondary follicle densities were significantly lower in lambs exposed to exogenous T4 compared with other treatments (P < 0.05). However, the T4 group displayed a higher proportion of mature secondary follicles (reflected by increased mature secondary follicle densities and mature secondary/primary follicle ratios) than the other treatment groups (P < 0.05). The skin morphology of the lambs differed 12 months later, with the T4 group having significantly higher total follicle densities compared with the PTU group, largely attributed to increased mature and total secondary follicle densities. However, this increase in wool follicle densities did not translate to differences in the fleece yields and weight, fibre diameter, staple lengths or any other fibre parameters. This study showed that transient manipulation of thyroid hormone status during foetal primary follicle initiation does have long-term consequences on the morphology of wool follicles, in particular the maturity of secondary wool follicles.

Wool fibre crimp is determined by mitotic asymmetry and position of final keratinisation and not ortho- and para-cortical cell segmentation.

Crimp, a distinguishing feature of sheep fibres, significantly affects wool value, processing and final fabric attributes. Several explanations for fibre bending have been proposed. Most concentrate on relative differences in the physicochemical properties of the cortical cells, which comprise the bulk of the fibre. However, the associations between cortical properties and fibre crimp are not consistent and may not reflect the underlying causation of fibre curvature (FC). We have formulated a mechanistic model in which fibre shape is dictated primarily by the degree of asymmetry in cell supply from the follicle bulb, and the point at which keratinisation is completed within the follicle. If this hypothesis is correct, one would anticipate that most variations in fibre crimp would be accounted for by quantitative differences in both the degree of mitotic asymmetry in follicle bulbs and the distance from the bulb to the point at which keratinisation is completed. To test this hypothesis, we took skin biopsies from Merino sheep from sites producing wool differing widely in fibre crimp frequency and FC. Mitotic asymmetry in follicle bulbs was measured using a DNA-labelling technique and the site of final keratinisation was defined by picric acid staining of the fibre. The proportion of para- to ortho-cortical cell area was determined in the cross-sections of fibres within biopsy samples. Mitotic asymmetry in the follicle bulb accounted for 0.64 (P < 0.0001) of the total variance in objectively measured FC, while the point of final keratinisation of the fibre accounted for an additional 0.05 (P < 0.05) of the variance. There was no association between ortho- to para-cortical cell ratio and FC. FC was positively associated with a subjective follicle curvature score (P < 0.01). We conclude that fibre crimp is caused predominantly by asymmetric cell division in follicles that are highly curved. Differential pressures exerted by the subsequent asymmetric cell supply and cell hardening in the lower follicle cause fibre bending. The extent of bending is then modulated by the point at which keratinisation is completed; later hardening means the fibre remains pliable for longer, thereby reducing the pressure differential and reducing fibre bending. This means that even highly asymmetric follicles may produce a straight fibre if keratinisation is sufficiently delayed, as is the case in deficiencies of zinc and copper, or when keratinisation is perturbed by transgenesis. The model presented here can account for the many variations in fibre shape found in mammals.

Use of statistical process control charts in stroke medicine to determine if clinical evidence and changes in service delivery were associated with improvements in the quality of care.

BACKGROUND AND OBJECTIVE: Monitoring the effect of service changes on quality of care is essential. By using statistical process control (SPC) charts, this study aimed to explore the relationship between changes in the structure of stroke services and the process of care. METHODS: Prospectively acquired data on the process of acute stroke care from three hospitals admitting 2962 patients (July 2001 to June 2004) were charted retrospectively on SPC charts for individual values (I charts) to determine whether or not "special cause variation" followed known changes in stroke service structure and publication of the Medical Research Council (MRC) Heart Protection Study. Unexpected signals of special cause variation were identified and reasons for observed patterns were sought by discussion with clinical teams. RESULTS: Improved brain imaging provision was followed by a reduction in time to imaging and earlier prescription of aspirin for ischaemic stroke. The MRC Heart Protection Study was followed by increased statin prescription. However, increasing beds allocated to stroke had no influence on the proportion of patients receiving stroke unit care. Some unexpected signals of special cause variation could be plausibly explained (eg, breakdown of brain scanner), but others could not. Anecdotal evidence from healthcare professionals suggests that charts may be acceptable in clinical practice. CONCLUSION: SPC charts have the potential to provide valuable insights into the impact of changes in structure of services and of clinical evidence on the process of stroke care. In the present study, the charts were generally well received by healthcare professionals.

The role of activins and follistatins in skin and hair follicle development and function.

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFbeta superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFbeta signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.

Adjusting proportional mortality ratios for the influence of extraneous causes of death.

Proportional mortality ratios (PMRs) are frequently used where denominators of the population at risk are not available or are known to be seriously biased. The interpretation of proportional measures requires care and can be complicated by the fact that a PMR of a cause of interest may be affected by disproportionate mortality from one or more other causes of death. This problem is examined using occupational mortality data for England and Wales. A method of allowing for the influence of other causes of death on a PMR of interest is proposed and it is argued that this approach is a useful aid to the general interpretation of PMRs.