bluuebunny/crossref_metadata_2025 huggingface dataset:
A stochastic model for the spatial‐temporal simulation of nonhomogeneous rainfall occurrence and amounts
Authors: Burton A. , Fowler H. J. , Kilsby C. G. , O'Connell P. E.
Published: November 2010

The nonhomogeneous spatial activation of raincells (NSAR) model is presented which provides a continuous spatial‐temporal stochastic simulation of rainfall exhibiting spatial nonstationarity in both amounts and occurrence. Spatial nonstationarity of simulated rainfall is important for hydrological modeling of mountainous catchments where orographic effects on precipitation are strong. Such simulated rainfall fields support the current trend toward distributed hydrological modeling. The NSAR model extends the Spatial Temporal Neyman‐Scott Rectangular Pulses (STNSRP) model, which has a homogeneous occurrence process, by generating raincells with a spatially nonhomogeneous Poisson process. An algorithm to simulate nonhomogeneous raincell occurrence is devised. This utilizes a new efficient and accurate algorithm to simulate raincells from an infinite 2‐D Poisson process, in which only raincells relevant to the application are simulated. A 4009 km2 Pyrenean catchment exhibiting extreme orographic effects provides a suitable case study comprising seven daily rain gauge records with hourly properties estimated using regional downscaling relationships. Both the NSAR and the STNSRP models are fitted to five calibration rain gauges. Simulated hourly fields are validated using the remaining two rain gauges providing the first validation of time series sampled from STNSRP or NSAR processes at locations not used in model fitting. The NSAR model exhibits considerable improvement over the STNSRP model particularly with respect to nonhomogeneous rainfall occurrence at both daily and hourly resolutions. Further, the NSAR simulation provides an excellent match to the spatially nonhomogeneous observed daily mean, proportion dry, variance, coefficient of variation, autocorrelation, skewness coefficient, cross correlation and extremes, and to the hourly proportion dry and variance properties.
Strong longitudinal variations in the OH nightglow
Authors: Xu Jiyao , Smith A. K. , Jiang Guoying , Gao Hong , Wei Yuan , Mlynczak M. G. , Russell J. M.
Published: November 2010

Airglow from the hydroxyl Meinel bands, originating from about 87 km, gives a signature of the atmosphere that can be observed remotely. Analysis of long term global observations of the 2.0 μm OH Meinel brightness observed by the TIMED/SABER satellite instrument presents some striking patterns that appear in the Meinel airglow. The analysis shows that migrating and non‐migrating tides have large effects on the nighttime OH airglow emission in the upper mesosphere. The OH airglow emission rate is positively correlated with temperature below 94 km and negatively correlated above. Variations with longitudinal wavenumbers 1 and 4 are shown to result from the impacts of the stationary (D0), westward wavenumber 2 (DW2), and eastward wavenumber 3 (DE3) nonmigrating diurnal tides.
Proteochemometric modeling as a tool to design selective compounds and for extrapolating to novel targets
Authors: van Westen Gerard J. P. , Wegner Jörg K. , IJzerman Adriaan P. , van Vlijmen Herman W. T. , Bender A.
Published: November 2010

Proteochemometric modeling is founded on the principles of QSAR but is able to benefit from additional information in model training due to the inclusion of target information.
Relations Between the Voice and the Ear With Clinical Implications
Authors: Hunter Eric J. , Monson Brian B. , Montequin Doug W.
Published: November 2010

The ear and the vocal mechanism are closely tied. We develop our voice and speech skills with constant aural feedback by making on-line changes in our acoustic output based on our self-perceived acoustic output. The efficacy of the human voice in communicating messages and ideas to others is inevitably dependent upon the human auditory system (of both the listener and the talker). Thus, a basic understanding of the relation of these two mechanisms is fundamental to an accurate assessment of potential vocal issues. The authors will review and discuss some of the relationships between the voice and the ear, the environmental factors that can affect aural feedback in a clinical setting, and the significance of this discussion to the speech-language pathologist.
Nonlinear reaction coordinate analysis in the reweighted path ensemble
Authors: Lechner Wolfgang , Rogal Jutta , Juraszek Jarek , Ensing Bernd , Bolhuis Peter G.
Published: November 2010

We present a flexible nonlinear reaction coordinate analysis method for the transition path ensemble based on the likelihood maximization approach developed by Peters and Trout [J. Chem. Phys. 125, 054108 (2006)]. By parametrizing the reaction coordinate by a string of images in a collective variable space, we can optimize the likelihood that the string correctly models the committor data obtained from a path sampling simulation. The collective variable space with the maximum likelihood is considered to contain the best description of the reaction. The use of the reweighted path ensemble [J. Rogal et al., J. Chem. Phys. 133, 174109 (2010)] allows a complete reaction coordinate description from the initial to the final state. We illustrate the method on a z-shaped two-dimensional potential. While developed for use with path sampling, this analysis method can also be applied to regular molecular dynamics trajectories.
Evidence for a simple monatomic ideal glass former: The thermodynamic glass transition from a stable liquid phase
Authors: Elenius Måns , Oppelstrup Tomas , Dzugutov Mikhail
Published: November 2010

Under cooling, a liquid can undergo a transition to the glassy state either as a result of a continuous slowing down or by a first-order polyamorphous phase transition. The second scenario has so far always been observed in a metastable liquid domain below the melting point where crystalline nucleation interfered with the glass formation. We report the first observation of the liquid-glass transition by a first-order polyamorphous phase transition from the equilibrium stable liquid phase. The observation was made in a molecular dynamics simulation of a one-component system with a model metallic pair potential. In this way, the model, demonstrating the thermodynamic glass transition from a stable liquid phase, may be regarded as a candidate for a simple monatomic ideal glass former. This observation is of conceptual importance in the context of continuing attempts to resolve the long-standing Kauzmann paradox. The possibility of a thermodynamic glass transition from an equilibrium melt in a metallic system also indicates a new strategy for the development of bulk metallic glass-forming alloys.
Hydrogen-induced change in the electrical properties of metal-insulator-semiconductor Pt–GaN diodes
Authors: Irokawa Yoshihiro
Published: November 2010

Exposure of Pt–SiO2–GaN diodes to hydrogen at room temperature is found to change the conduction mechanisms from Fowler–Nordheim tunneling to Pool–Frenkel emission. The capacitance-voltage (C-V) curve for Pt–SiO2–GaN diodes in hydrogen significantly shifts toward negative bias values as compared with that in nitrogen. In sharp contrast, Pt–SixNy–GaN diodes exhibit Pool–Frenkel emission in nitrogen and do not show any change in the conduction mechanism upon exposure to hydrogen. The C-V curve for Pt–SixNy–GaN diodes also does not show any shifts upon the exposure to hydrogen. These results suggest that the work function change in the Schottky metal is not responsible mechanism for the hydrogen sensitivity. The interface between the metal and the semiconductor plays a critical role in the interaction of hydrogen with semiconductor devices, including diodes and field-effect transistors (FETs).
Laser properties of the diode-pumped Nd3+-doped La2CaB10O19 crystal
Authors: Brenier A. , Wu Y. , Zhang J. , Wu Y. , Fu P.
Published: November 2010

We have determined the angular distribution of the fluorescence at 1051 nm wavelength of the Nd3+-doped monoclinic La2CaB10O19 crystal, originating from Nd3+ ions located in the regular La3+ sites, for extraordinary polarization in the XZ principal plane. We have found that the highest emission intensity does not occurs the Z-axis direction but in a −7.1° around the Y=b-axis rotated direction. With a one-pump cavity we obtained simultaneous lasing at 1051 and 1069 nm in X and Z-polarizations from Nd3+ ions located in La3+ and Ca2+ sites, respectively. In the active Q-switch regime the two laser pulses are always simultaneous. With a two-pump setup based on an optical bifurcated fiber we obtained also simultaneous lasing at 1051 and 1069 nm. The relative power of the two laser channels is tuneable with the help of a beam-splitter. An adequate value of the relative pump power of the two channels leads to simultaneous pulses in the active Q-switch regime.
Efficient parametric amplification in high and very high frequency piezoelectric nanoelectromechanical systems
Authors: Karabalin R. B. , Masmanidis S. C. , Roukes M. L.
Published: November 2010

Parametric amplification in nanomechanical structures is demonstrated by modulating a purely intrinsic mechanical parameter of the system—the stress—via piezoelectric electromechanical coupling. Large resonance amplitude and quality factor enhancement due to parametric pumping are observed under both vacuum and ambient pressure conditions. Exploration of the region of parametric instability yields results that agree with parametric amplification theory.
Ultrafast carrier and phonon dynamics in Bi2Se3 crystals
Authors: Qi J. , Chen X. , Yu W. , Cadden-Zimansky P. , Smirnov D. , Tolk N. H. , Miotkowski I. , Cao H. , Chen Y. P. , Wu Y. , Qiao S. , Jiang Z.
Published: November 2010

Ultrafast time-resolved differential reflectivity of Bi2Se3 crystals is studied using optical pump-probe spectroscopy. Three distinct relaxation processes are found to contribute to the initial transient reflectivity changes. The deduced relaxation timescale and the sign of the reflectivity change suggest that electron–phonon interactions and defect-induced charge trapping are the underlying mechanisms for the three processes. After the crystal is exposed to air, the relative strength of these processes is altered and becomes strongly dependent on the excitation photon energy.
The physics of boron nitride nanotubes
Authors: Cohen Marvin L. , Zettl Alex
Published: November 2010

As synthesis techniques mature, BN nanotubes are likely to be increasingly valuable in fundamental physics studies and find application in a variety of forms—from dyeable, neutron-absorbing fabrics to high-temperature catalysts and photovoltaic devices.
004.From molecules to mind: stress, allostasis and integration of brain and body
Authors: McEwen B.
Published: October 2010

The mind involves the whole body and two-way communication between the brain and the cardiovascular, immune and other systems via neural and endocrine mechanisms. Stress is a condition of the mind and a factor in the expression of disease that differs among individuals. A broader view is that it is not just the dramatic stressful events that exact their toll but rather the many events of daily life that elevates activities of physiological systems so as to cause some measure of wear and tear. We call this wear and tear 'allostatic load', and it reflects not only the impact of life experiences but also genetic load' individual life-style habits reflecting items such as diet, exercise and substance abuse' and developmental experiences that set life-long patterns of behavior and physiological reactivity (1).
Hormones associated with stress and allostatic load protect the body in the short-run and promote adaptation, but the long run allostatic load causes changes in the body that lead to disease. This will be illustrated for the immune system and brain regions involved in stress, fear and cognition (e.g. hippocampus, amygdala and prefrontal cortex). Besides developmental influences associated with mother–infant interactions, the most potent of stressors in adult life are those arising from competitive interactions between animals of the same species, leading to the formation of dominance hierarchies. Psychosocial stress of this type not only impairs cognitive function of lower ranking animals, but it can also promote disease (e.g. atherosclerosis) among those vying for the dominant position, as well as depressive illness. Social ordering in human society is also associated with gradients of disease, with an increasing frequency or mortality and morbidity as one descends the scale of socioeconomic status (SES) that reflects both income and education. Although the causes of these gradients of health are very complex, they are likely to reflect, with increasing frequency at the lower end of the scale, the cumulative burden of coping with limited resources and negative life events as well as differences in life style, and the allostatic load that this burden places on the physiological systems involved in adaptation and coping.
(1) McEwen, B.S. (1998) Protective and damaging effects of stress mediators. New England J. Med. 238, 171–179.
012.Metabolic determinants of implantation success and programming long term viability in embryos
Authors: Thompson J. G. , Kind K. L.
Published: October 2010

It has long been recognised that energy substrate supply and metabolism are key determinants of early embryo development during in vitro culture. Recently it has been revealed that exposure to suboptimal metabolic environments during early embryo development can 'programme' subsequent development, leading to perturbed fetal development. For example, amino acid uptake profiles during early cleavage have been found to predict subsequent embryo development and potentially implantation success. However, the by-product of amino acid metabolism, ammonium, has also been found to significantly alter development, possibly through perturbed methylation of imprinted genes. Our own work has focussed on the role of oxygen availability and subsequent embryo development. Somatic cells respond to changing oxygen concentration by altering intracellular REDOX state (the balance between oxidative and reductive power within a cell), which in turn can alter transcription via REDOX-sensitive transcription factor activity. Furthermore, oxygen is known to have direct effects on transcriptional activity via the hypoxia-inducible factors (HIFs), transcription factors whose stability and DNA-binding activity are directly regulated by pO2, in particular under hypoxic conditions. Using a mouse model, we have demonstrated that reducing pO2 from 50�mmHg to 15�mmHg during the compaction and blastulation periods alone significantly alters expression patterns of oxygen-sensitive genes (such as glucose transporters), without significantly altering developmental progression to the blastocyst stage. Following transfer, embryos cultured under 15�mmHg O2, despite similar implantation rates, produced fewer viable and lighter fetuses than in vivo-derived control embryos or those cultured in either atmospheric or 50�mmHg pO2. This demonstrates that mouse embryos are sensitive to changes in their metabolic state during the post compaction period and that operating through causal pathways, the environment during this period of development can significantly affect subsequent developmental potential. Ironically, bovine embryo development appears to benefit under a low O2 concentration. Furthermore, HIF protein stability appears to differ between the two species, which may be the underlying cause for the differences in gene expression and developmental competence.
022.Spermatogonial stem cells: from basic research to clinical applications
Authors: Schlatt S.
Published: October 2010

The testis contains undifferentiated spermatogonia and is therefore the only adult organ populated with proliferating germline cells. Whereas the biology of these cells is quite well understood in rodents, their modes of mitotic expansion and differentiation are poorly understood in primates. The existence of these cells offers clinically relevant options for preservation and restoration of male fertility. New approaches based on male germ cell transplantation and testicular tissue grafting can be applied to generate a limited number of sperm and could therefore be considered as important new avenues applicable to a variety of disciplines like animal conservation, genetic germline modification or restoration of fertility in oncological patients. In principle, germ cell transplantation presents a removal of the stem cell from the donor's niche and a transfer into the niche of a recipient. Grafting can be considered as a transplantation of the stem cell in conjunction with its niche. Germ cell transplantation of human spermatogonia into mouse testes revealed that the stem cells survive and expand but are not able to differentiate and complete spermatogenesis. We have developed an approach to infuse germ cells into monkey and human testes and showed that germ cell transplantation is feasible as an autologous approach in primates. Furthermore, we applied germ cell transplantation in the monkey model mimicking a gonadal protection strategy for oncological patients. Ectopic xenografting of testicular tissue was applied to generate fertile sperm from a variety of species. Newborn testicular tissue was grafted into the back skin of immunodeficient mice and developed up to qualitatively complete spermatogenesis. The rapid progress in the development of novel experimental strategies to generate sperm from cryopreserved spermatogonial stem cells or immature testicular tissue will lead to many new options for germline manipulation and fertility preservation.
023.Drivers of germ cell differentiation
Authors: Loveland K. L. , Hogarth C. , Szczepny A. , Itman C. , Huebner A. , Jans D. A.
Published: October 2010

Spermatogenesis requires progression of a self-renewing male germline stem cell population through a precisely timed and ordered developmental sequence to form spermatozoa. For several years, we have been investigating the functional impact of signals on this progression by members of the TGFβ superfamily, follicle stimulating hormone and the bcl-2 family. For example, our lab and others have shown that activin A, bone morphogenetic protein (BMP)-4 and glial-derived neurotropic factor (GDNF) all modulate stem cell development and spermatogonial differentiation at the onset of spermatogenesis after birth. We are trying to understand how germ cells 'interpret' this plethora of competing signals to mediate maturation.
Progression through successive maturation states in response to such signals requires the movement of proteins, including transcription factors, into the nucleus to implement changes in gene transcription. We are investigating the concept that regulated transport of proteins into the nucleus is one mechanism that governs spermatogenic differentiation, and we have focused on analysis of the major class of nuclear transport factors, the importins (IMPs). A diverse family comprising at least six different α forms and 20 different β forms in the mouse, the IMP proteins selectively bind a diverse range of cargo proteins and mediate their passage through the nuclear pore complex and into the nucleus. Our immunohistochemical and in situ hybridisation studies have demonstrated developmentally regulated expression of many IMPs in germ cells, in the fetus, in the neonate and in the adult. This suggests that they function to transport cargo required for discrete stages of spermatogenesis. Our recent studies examined the mouse embryonic gonad around the time when specification to form either a testis or an ovary occurs. The IMPβ3 protein is present in both male and female germ cells, but the subcellular localisation and expression patterns within these cell types is gender- and age-specific. We are currently exploring the functional significance of our observations.
025.Ovaries: up in a POF of smoke
Authors: Shelling A. N. , Smale W. , Prendergast D. , Harris S. E. , Chand A. L. , Ramachandran A. , Woad K. , Winship I. M.
Published: October 2010

Premature ovarian failure (POF) or premature menopause is a common disorder, defined by the occurrence of menopause under the age of 40 years and is characterised by amenorrhoea, hypoestrogenism and elevated gonadotrophins. Worldwide it affects 1% of all women and occurs in 0.1% before the age of 30 years. The major problems associated with POF are the loss of fertility at an early age and the psychological problems associated with this. In addition there are the physiological effects of reduced oestrogen, which include an increased risk of osteoporosis. POF is a heterogeneous disorder and the cause of most cases is unknown. A significant proportion (20-30%) of women with POF have a genetic predisposition. Our primary goal is to identify genes involved in POF. In most cases, the menopause is due to the loss of follicles, and it stands to reason that suitable candidate genes for POF development would be genes that regulate the rate of follicle loss. We have identified two common gene mutations, a 769G>A transition in the inhibin alpha gene in approximately 5% of POF patients associated with POF at a very early age, and mutations in FOXL2 in approximately 5% of patients. FOXL2 is thought to act downstream of inhibin which suggests that other candidate genes may arise from the analysis of the activin signalling pathway. Functional studies will help us to understand more about the molecular basis of POF. Each mutation has been associated with less than 10% of POF cases and POF is likely to be caused by mutations at many different loci. It is hoped that determining the molecular basis of POF will lead to the development of genetic tests to predict the development of POF, and eventually lead to treatment that will return fertility to these women.
042.The role of TGF-β in normal and pathological lens development
Authors: McAvoy J. W. , Lovicu F. J. , De Iongh R. U.
Published: October 2010

How the lens develops its highly ordered architecture and growth patterns is a major question in developmental biology. During embryogenesis, cells in the anterior and posterior segments of the lens vesicle, differentiate into the epithelial and fibre cells, respectively. Our research has aimed to identify the molecules and mechanisms that regulate the divergent fates of lens cells. We have studied the roles of various growth factors in regulating lens cell fates using rat lens epithelial explant cultures and transgenic and mutant mouse models. Our research has shown that members of the FGF growth factor family are key initiators of lens fibre differentiation in mammals and there is now compelling evidence that a gradient of FGF in the eye controls lens polarity and growth patterns. Recent evidence also supports a role for TGF-β signalling in this process and indicates that a cascade of growth factor signalling may be required for normal fibre differentiation. Less is known about the anterior segment; however, our recent studies point to an important role for the Wnt growth factor family in epithelial differentiation. Growth factor signalling can also cause pathological changes; e.g. TGF-β can destabilise the normal epithelial phenotype and induce aberrant growth and differentiation that mimics the epithelial-mesenchymal transition characteristic of some forms of cataract. These studies highlight the importance of growth factor signalling in regulating the ordered growth and differentiation of the lens. It is also clear that the bioavailabity of some growth factors needs to be tightly regulated so that they act in the appropriate cellular compartment. Some cataracts may be a consequence of disturbed growth factor, particularly TGF-β, regulatory mechanisms.
159. Polycystic ovary syndrome: are endocrinologists and gynecologists treating the same patients?
Authors: Cussons A. J. , Stuckey B. G. A. , Walsh J. P. , Burke V. , Norman R. J.
Published: October 2010

Women with polycystic ovary syndrome commonly consult endocrinologists or gynaecologists. The diagnosis and management of this disorder are controversial, and it is not known if these specialty groups differ in their approach. Our objective was to compare the investigation, diagnosis and treatment of polycystic ovary syndrome by endocrinologists and gynaecologists. A questionnaire containing a hypothetical patient case history with varying presentations was sent to endocrinologists and gynaecologists in teaching hospitals and private practice. Evaluable responses were obtained from 138 endocrinologists and 172 gynaecologists. The two specialty groups differed markedly in their choice of essential diagnostic criteria. Endocrinologists regarded androgenisation (81%) and menstrual irregularity (70%) as essential for diagnosis, whereas gynaecologists cited polycystic ovaries on ultrasound (61%), androgenisation (59%), menstrual irregularity (47%) and elevated LH�:�FSH ratio (47%). (All P values <0.001.) Gynaecologists were more likely to request ovarian ultrasound (91% v. 44%, P�<�0.001) whereas endocrinologists were more likely to measure adrenal androgens (80% v. 58%, P�<�0.001) and fasting lipids (67% v. 34%, P�<�0.001). Gynaecologists were less likely to assess glucose homeostasis but were more likely to use a glucose tolerance test to do so. Diet and exercise were chosen by most respondents as first-line treatment for oligomenorrhoea, hirsutism, infertility and obesity. Endocrinologists were more likely to use insulin sensitisers, particularly metformin, for these indications. In particular, for infertility, endocrinologists favoured metformin treatment whereas gynaecologists recommended clomiphene. There is a lack of consensus between endocrinologists and gynaecologists in the definition, diagnosis and treatment of polycystic ovary syndrome. Women may receive different diagnostic advice and treatment depending on the type of specialist consulted.
160.Ovarian leukocytes and cytokines in polycystic ovary syndrome
Authors: Norman R. J. , Wu R. , Fujii S. , Ryan N. , Van Der Hoek K. , Jasper M. , Sini I.
Published: October 2010

Polycystic ovary syndrome (PCOS) is associated with anovulatory infertility and metabolic disturbances. PCOS has an unknown aetiology but is involved with aberration of substances that lead to follicular growth. The ovarian leukocyte/cytokine network is important in the ovary and has not been adequately examined in PCOS. The aim of the study was to look at the distribution of leukocytes in the ovaries of women with PCOS and to look at expression of cytokine and chemokine mRNA in follicular cells from these patients. Ovaries were obtained from PCOS (n�=�5) and non-PCOS (n�=�4) women undergoing gynaecological surgery for non-ovarian conditions prior to the menopause. They were immunostained for a wide variety of leukocyte markers and distribution counted using visual imaging software. Luteinising granulosa cells were obtained prior to ovulation in women undergoing in vitro fertilization with (n�=�11) and without (n�=�22) PCOS and mRNA studied using quantitative RT-PCR for various cytokines and chemokines. The CD45RO subset of leukocytes (principally activated/memory T-lymphocytes) were significantly decreased in ovaries from PCOS women compared to non-PCOS women. The other leukocytes were not different in distribution and numbers. Transcripts for CSF-1, IL-1β, IL-6, IL-8, IL-10, MCP-1 and TNFα were not different between PCOS and non-PCOS women whilst GM-CSF mRNA was not detectable in either group. There was an association between high testosterone levels and high IL1β and low TNFα transcripts. Women who became pregnant following IVF had higher levels of IL-10 mRNA. The role of T-lymphocytes in PCOS needs further examination, and if the leukocyte/cytokine network in PCOS is important, other cells and cytokines need examination. This is the first study to definitively describe the leukocyte/cytokine network within polycystic ovaries. While other cells and substances may be important in PCOS and intervention procedures such as ovarian drilling, it does not appear as though macrophages, neutrophils, B-lymphocytes and a variety of cytokines are involved in the aetiology of PCOS.
204.Regulation of bovine oocyte meiotic and developmental capacity by glucose and glucosamine
Authors: McDowall M. L. Sutton , Gilchrist R. B. , Thompson J. G.
Published: October 2010

Glucose is an important substrate for in vitro oocyte maturation (IVM) and is metabolised by cumulus oocyte complexes (COCs) via glycolysis or is used for extracellular matrix (ECM) synthesis. Follicular glucose concentration is significantly lower than commonly used IVM media (2.3�mM v. 5.6�mM in TCM199). Glucosamine is an alternative substrate for ECM and supplementation to IVM media reduces glucose uptake by COCs. The aim of this study was to determine the effect of glucose and glucosamine supplementation during IVM on bovine oocytes. First, bovine COCs (n�=�400) were matured in TCM199 (containing pyruvate, BSA, hCG and FSH), or synthetic follicular fluid medium (SFFM; a defined medium based on bovine follicular fluid composition) with 2.3�mM or 5.6�mM glucose���5�mM glucosamine and nuclear maturation was assessed after 24 and 30�h. Significantly less COCs matured in 2.3�mM glucose completed nuclear maturation compared to COCs matured in 5.6�mM glucose (P�<�0.05), whereas glucosamine had no effect on meiotic maturation. We then compared oocyte developmental capacity following IVM (n�=�600) in TCM199 or SFFM�+�5.6�mM glucose���5mM glucosamine. Blastocyst production was severely perturbed when COCs were matured in the presence of glucosamine (–glucosamine 32% v. +glucosamine 4%; P�<�0.001). To determine the cause of this reduction in oocyte developmental competence, we investigated oocyte protein synthesis by maturing COCs (n�=�100) in SFFM�+�5.6�mM glucose���5mM glucosamine�+�1�mM L-[2,3,4,5,6–3H] phenylalanine. In the presence of glucosamine, oocyte protein synthesis was reduced 40% compared to oocytes matured in control medium (P�<�0.05).
These results demonstrate that while glucosamine supplementation has no effect on oocyte nuclear maturation, cytoplasmic maturation is compromised, as demonstrated by perturbed oocyte protein synthesis and embryo development. In contrast, glucose concentration has a significant influence on meiotic progression. This provides a useful model to investigate the mechanisms of establishment of developmental competence in oocytes following maturation.
206.Cell death of the theca interna during bovine ovarian follicular atresia
Authors: Clark L. J. , Irving-Rodgers H. F. , Dharmarajan A. M. , Rodgers R. J.
Published: October 2010

It is generally accepted that death of cells within the theca interna occurs late during ovarian follicular atresia. Histological classifications of atresia are usually based solely upon the morphology of the membrana granulosa. Atresia of bovine antral follicles less than 5 mm in diameter has been redefined as either antral or basal atresia depending on where in the membrana granulosa cell death is initiated. The aim of present study was to investigate changes within the theca interna during both antral and basal atresia. Bovine ovaries were collected and processed for light microscopy and immunohistochemistry. Each follicle less then 5 mm was classified as either healthy, antral atretic or basal atretic, with antral atresia being further classified either early-mid or late stage. Sections were labelled by TUNEL to identify dead cells combined with lectin from Bandeiraea simplificifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles compared to healthy and antral atretic follicles. In both antral and basal atresia there was death of endothelial cells and steroidogenic cells. However cell death was greater in endothelial cells (P < 0.05) and steroidogenic cells (P < 0.001) of the theca interna of basal atretic follicles. There was no significant difference in the amount of cell death in the membrana granulosa between early-mid antral atresia and basal atresia while death of the membrana granulosa was significantly increased in late antral atresia compared to basal atresia (P < 0.01). Therefore we conclude that basal atresia is not a progression of antral atresia and that the theca interna can be susceptible to cell death early in atresia in basal atretic follicles.
214.Identification of potential zona pellucida-binding sperm proteins using heat shock protein 60
Authors: Walsh A. K. , Asquith K. L. , Thomas C. M. , Mitchell L. A. , Nixon B. , Aitken R. J.
Published: October 2010

Sperm transit through the female tract is correlated with a number of biochemical and physical changes, termed capacitation, which culminate in acquisition of the ability to fertilise an egg. One of the important correlates of capacitation is an increase in tyrosine phosphorylation of multiple sperm proteins. Recent studies of the sperm surface proteome have demonstrated that, following capacitation, a number of tyrosine-phosphorylated proteins become expressed on the sperm surface. Analysing these proteins, we have identified at least three members of the chaperone family including heat shock protein 60 (HSP60), heat shock protein 90 (HSP90) and Endoplasmin (ERP99). Although these proteins are not directly implicated in zona binding, we hypothesise that they are involved in the assembly of a multimeric zona receptor on the sperm surface. To investigate this hypothesis, we have initiated studies to purify sperm surface proteins which associate with these chaperones. This has involved the cross-linking of sperm surface proteins, using a membrane impermeable cross-linker, followed by extraction of the surface membrane proteins. Co-immunoprecitation of these cross-linked surface proteins from capacitated sperm has yielded a number of proteins associated with the chaperone HSP60. The characterisation of these proteins is the focus of ongoing research.
217.Expression of the chemokine CXCL12 and its receptor CXCR4 in the activating mammalian ovarian follicle
Authors: Holt J. E. , Aitken R. J. , Roman S. S. , McLaughlin E. A.
Published: October 2010

The mammalian ovary contains a finite number of oocytes that is determined during oogenesis in fetal life. As most of these oocytes are destined to undergo apoptosis, the initial primordial follicle population represents a valuable resource for the clinical manipulation of the female germ cell pool. However the events underlying the activation of the resting primordial follicle remain relatively poorly understood. A comparison was undertaken between whole 2-day and 7-day neonate mouse ovaries which represents ovaries with primordial follicles and primordial follicles / newly activated follicles respectively. The comparison took place at the level of gene expression utilizing cDNA microarray analysis. The mRNAs for the chemokine CXCL12 and its receptor CXCR4, were consistently shown to be up-regulated approximately 2-fold in 7-day tissue compared with 2-day tissue. Microarray results were confirmed for CXCL12 by real-time PCR analysis. CXCL12 and CXCR4 have been identified as essential for development of the haemopoietic, nervous and cardiovascular systems and have also been shown to be involved in the foetal migration of primordial germ cells to the gonads. These genes were selected for further analysis due to the known importance of other cytokine family members in primordial follicle activation. In situ hybridisation studies of CXCL12 revealed mRNA expression in oocytes of all stages, including primordial follicles, as well as epithelial, corpora luteal and stromal cells. In contrast CXCR4 mRNA expression appears to be restricted primarily to oocytes of all stages. This co-expression of ligand and receptor within the oocyte suggests an autocrine signaling mechanism. The age-dependent increase in CXCL12 and CXCR4 gene expression is likely to be a result of an increased oocyte cytoplasmic volume and/or the proportion of stromal cells present as follicles begin to activate in the neonate ovary. Examination of the timing of protein expression is currently underway to identify the role this chemokine signaling pathway plays in the initial activation of the mammalian primordial follicle.
218.FGF9 stimulates ovarian progesterone production
Authors: Drummond A. E. , Dyson M. , Findlay J. K.
Published: October 2010

FGF9, a member of the fibroblast growth factor family (FGF), is known to be a male sex-determining factor involved in testicular cord formation (1). FGF9 knockout males are sex-reversed (2). However, nothing is known about FGF9's role in folliculogenesis because these mice die at birth (2). We previously reported the presence of FGF9 mRNA and protein in the immature rat ovary (3). In these studies we investigated: (1) the presence of FGF9 receptors (FGFR3) on granulosa cells (GC); and (2) the impact of FGF9 on GC progesterone production.
GC isolated from 21 day old diethylstilboestrol (DES)-treated rats were cultured for either 2 hours (RNA) or 2 days (progesterone) in McCoys 5C with FGF9 (0.1-50ng/ml) � FSH (100ng/ml). Progesterone was measured in conditioned media by radioimmunoassay. RNA was extracted from the granulosa cells and reverse-transcribed for PCR. Specific primers for P450 side chain cleavage (SCC) amplified a 329�bp cDNA fragment. GAPDH was used for data normalisation. The FGF9 receptor FGFR3, was immunolocalised on formalin-fixed, paraffin-embedded sections of immature rat ovary.
FGFR3 protein was localised only to GC of the ovary. Progesterone production by cultured GC was significantly elevated by FGF9, consistent with the presence of FGFR3. Relative to a maximally stimulating dose of FSH, FGF9 increased progesterone production 10- fold. In preliminary studies, FGF9 increased the expression of P450 SCC mRNA by cultured GC revealing a mechanism by which FGF9 increases progesterone production. These data suggest a role for FGF9 not just in testicular formation, but in the regulation of ovarian steroidogenesis. Supported by the NH&MRC of Australia (Regkey 241000 & 198705).
(1) Cotinot et al. (2002) Semin. Reprod. Med. 20, 157. (2) Colvin et al. (2001) Cell 104, 875. (3) Drummond et al. (2003) SRB Abstract 90.
222.Characterisation of the infertility effect induced by a recombinant murine cytomegalovirus expressing murine zona pellucida 3
Authors: Lloyd M. L. , Papadimitriou J. M. , O'Leary S. , Lawson M. A. , Shellam G. R.
Published: October 2010

A recombinant mouse virus, murine cytomegalovirus, that has been engineered to encode the fertility antigen murine zona pellucida 3 (mZP3), is being developed for fertility control in mice. A single inoculation of the recombinant virus induces complete infertility in female BALB/c mice which persists for the breeding life of the animal. The extent of this autoimmune response was unexpected especially as the incorporation of mZP3 appears to have immunologically attenuated the growth of the recombinant virus. The histological features of the infection are an initial depletion in tertiary follicles by 21 days post inoculation followed by a progressive depletion of primordial follicles, leading to an almost complete absence of follicles by 150 days post-infection. High titre, long lasting, zona pellucida-specific antibody is present in infertile BALB/c mice although infertility has not been linked with either a critical titre or a dominant immunoglobulin isotype. However, our evidence suggests that anti-ZP3 antibody plays a primary role in infertility since antibodies are detected in vivo bound to the zona pellucida of ovaries from recombinant virus-infected mice, and passively transferred antibody from infected animals induces infertility in the absence of recombinant virus. In addition, an experiment in which immunoglobulin-deficient mice remained fertile after inoculation with the recombinant virus indicates that antibody is crucial for the immunocontraceptive effect to occur. Other immune mechanisms are also being explored.
223.Ovarian pathology in mice following immunisation with recombinant murine cytomegalovirus expressing murine zona pellucida 3
Authors: O'Leary S. , Lloyd M. L. , Shellam G. R. , Maddocks S.
Published: October 2010

Immunocontraception is a promising biological control for wild mice in Australia, having the potential to reduce the socioeconomic cost of plagues with minimal environmental impact. Inoculation of BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (mCMV-ZP3) confers total infertility characterised by depletion in ovarian tertiary follicles by Day 21 post inoculation followed by a progressive depletion in primordial follicles (1). The mechanisms underlying ovarian pathology are largely unknown but are likely to involve antibody mediated and cell mediated immune responses. The immune pathology may also be facilitated by acute responses involving antibody binding to ZP in growing follicles resulting in recruitment of inflammatory cells and oocyte destruction. The aim of this study was to investigate the effect of mCMV–ZP3 infection on leukocyte infiltration and expression of oocyte-derived signalling molecules in ovarian tissue. Fifteen BALB/c female mice were randomly allocated into three groups of 5 animals. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2�×�104�p.f.u. of mCMV and mCMV-ZP3 respectively. Ovaries were retrieved at Day 7 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages. The other ovary was processed for real time quantitative RT-PCR analysis of growth and differentiation factor 9 (GDF-9) and connexin 43 (Cx43) expression. mCMV-ZP3 inoculation increased the abundance of ovarian leukocytes (P�=�0.08), significantly increased expression of Cx43 mRNA (p<0.05), but did not alter GDF-9 mRNA expression. These results suggest that changes in expression of ovarian regulators due to ZP3 immunisation begins early after recombinant MCMV infection in mice, and implicates leukocyte infiltration in the mechanism leading to permanent ovarian failure. Further experiments are underway to investigate the dynamics of leukocyte trafficking and expression of oocyte-derived signals as the course of infection progresses. This study is funded by the Cooperative Research Centre for Pest Animal Control.
(1) Lloyd, M. L., et al. (2003). Biology of Reproduction 68(6): 2024-32.
230.Novel immune modulation to improve reproductive outcomes in pigs
Authors: Bray M. , O'Leary S. , Brooke G. , Maddocks S. , Armstrong D. T.
Published: October 2010

Early embryonic mortality is a major factor limiting litter size and profitability in the pig industry. Pregnancy success requires an immunologically receptive reproductive tract, generated in response to exposure to immune modulating factors in the seminal plasma (SP). Both the short-term pro-inflammatory response of the endometrium to SP and to a novel immune-modulating product, mycobacterial cell wall extract (MCWE; 'Equimune', Bioniche Animal Health) and the reproductive outcomes from a large-scale farrowing trial are described. In the first experiment, 15 Large White gilts (24 weeks of age) were randomly allocated to three intrauterine treatments administered at onset of gonadotrophin-induced oestrus: (1) 80 mL PBS (control); (2) 80 mL SP; or (3) 80 mL PBS containing 500 �g MCWE. Gilts were slaughtered 32–34 h later and reproductive tracts retrieved. Luminal fluid leukocytes were assessed following fixation using DIFF-Quik stain, and indicated the proportion of lymphocytes increased with SP and MCWE treatment compared to the control group (14.0% and 17.0% v. 9.3%; P < 0.05), and the proportion of monocytes decreased (12.3% and 15.0% v. 25.0%; P < 0.05). In the second experiment, Large White or Large White/Landrace crossbred females (n�=�161) were artificially inseminated twice, following standard industry practice, after detection of standing oestrus. Stratified for parity and breed, each was allocated to a treatment group: (a) Control: standard AI dose 1st and 2nd insemination; (b) 500 μg MCWE added to 2nd insemination; or (c) 500 μg MCWE included in the 1st and 2nd insemination. The overall pregnancy rate was 91%, with no significant effect of treatment on litter size, average piglet bodyweight at birth or litter variability. Although the use of MCWE at the time of AI did not improve reproductive outcome, significant potential remains in utilising its immune stimulating properties in 'priming' the reproductive tract, followed by mating at the next oestrus. This approach may improve gilt farrowing rates, thus increase overall herd productivity and efficiency.
233.Ammonium affects mitochondrial distribution and function in mouse 2-cell embryos
Authors: Zander D. L. , Froiland D. A. , Lane M.
Published: October 2010

Amino acids are key regulators of embryo function and are essential components in embryo culture media. Amino acids spontaneously breakdown and are metabolised by embryos resulting in ammonium build-up in the medium. While ammonium does not affect blastocyst development, the ability of these blastocysts to implant was reduced along with subsequent fetal growth rates. However, the mechanism for the inhibitory effect of ammonium is currently not known. It has been demonstrated in other tissues that mitochondrial bioenergetics can be disrupted by the presence of ammonium in the media which subsequently affects cellular viability. Therefore, the aim of this study was to examine the effects of ammonium on the mitochondria of mouse embryos cultured in the presence of ammonium. Mouse zygotes from superovulated females were cultured in medium G1.2 with or without 300 μM ammonium for 22 h at 37oC in 6%CO2�:�5%O2�:�89%N2. In vivo-developed 2-cell embryos were flushed from the reproductive tract and assessed immediately. At the 2-cell stage mitochondrial distribution (Mitotracker) and membrane potential (JC-1) were assessed using confocal microscopy and images were quantitated using IP Lab software package. Differences between treatments were determined using ANOVA and Bonferroni's multiple comparison procedure. Culture of zygotes to the 2-cell stage in medium G1.2 did not affect mitochondrial distribution compared to in vivo controls. However, 2-cell embryos cultured with ammonium had a decrease in their mitochondrial nuclear�:�cortical ratio (97���1 compared to 106���1; P�<�0.05) indicating that mitochondria were dispersing away from the nuclei. Culture with ammonium also significantly decreased the mitochondrial membrane potential (0.50���0.01 mean pixel intensity ratio) compared to those cultured without ammonium (0.72���0.3 mean pixel intensity ratio, P�<�0.001). The data presented demonstrates that culture for only 24�h with ammonium disrupts both mitochondrial distribution and membrane potential and supports our hypothesis that mitochondria are an early target for the inhibitory action of ammonium.