Comparative proteomic analysis to study molecular events during gonad development in mice

Sex determination represents a critical bifurcation in the road of embryonic development. It is based on a finely regulated network of gene activity, as well as protein-protein interactions and activation or silencing of signaling pathways. Despite the identification of a number of critical genes, many aspects of the molecular cascade that drives the differentiation of the embryonic gonad into either a testis or an ovary remain poorly understood. To identify new proteins involved in this cascade, we employed two-dimensional gel electrophoresis and mass spectrometry to compare the protein expression profiles of fetal mouse testes and ovaries. Three proteins, hnRPA1, TRA1, and HSC71, were found to be expressed in a male-specific manner and this expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. Moreover, HSC71 was found to be hyperphosphorylated in male compared to female gonads, emphasizing the advantage of the proteomic approach in allowing the detection of posttranslational modifications.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-genii cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool. (c) 2006 Elsevier Inc. All rights reserved.

Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes

Despite the identification of SRY as the testis-determining gene in mammals, the genetic interactions controlling the earliest steps of male sex determination remain poorly understood. In particular, the molecular lesions underlying a high proportion of human XY gonadal dysgenesis, XX maleness and XX true hermaphroditism remain undiscovered. A number of screens have identified candidate genes whose expression is modulated during testis or ovary differentiation in mice, but these screens have used whole gonads, consisting of multiple cell types, or stages of gonadal development well beyond the time of sex determination. We describe here a novel reporter mouse line that expresses enhanced green fluorescent protein under the control of an Sf1 promoter fragment, marking Sertoli and granulosa cell precursors during the critical period of sex determination. These cells were purified from gonads of male and female transgenic embryos at 10.5 dpc (shortly after Sry transcription is activated) and 11.5 dpc (when Sox9 transcription begins), and their transcriptomes analysed using Affymetrix genome arrays. We identified 266 genes, including Dhh, Fgf9 and Ptgds, that were upregulated and 50 genes that were downregulated in 11.5 dpc male somatic gonad cells only, and 242 genes, including Fst, that were upregulated in 11.5 dpc female somatic gonad cells only. The majority of these genes are novel genes that lack identifiable homology, and several human orthologues were found to map to chromosomal loci implicated in disorders of sexual development. These genes represent an important resource with which to piece together the earliest steps of sex determination and gonad development, and provide new candidates for mutation searching in human sexual dysgenesis syndromes.

Stephanostomum talakitok n. sp (Digenea : Acanthocolpidae) from the golden trevalley, Gnathanodon speciosus (Perciformes : Carangidae), from Ningaloo Reef, Western Australia

A new species, Stephanostomum talakitok n. sp., is described from the golden trevally Gnathanodon speciosus, Ningaloo Reef, Western Australia. It has 36 (34-40) circum-oral spines and the vitellarium reaches to no less than 10-17% of the hindbody length from the ventral sucker. It differs from other species of Stephanostomum with these characteristics by combinations of the gradual diminution of the circum-oral spine size to a small mid-ventral spine, the contiguous gonads with no intervening vitelline follicles, the sucker-ratio and various other ratios, including the distance between the ventral sucker and the ovary and the distance the cirrus-sac reaches into the hindbody, both as a proportion of body length.

The number, morphology, and distribution of retinal ganglion cells and optic axons in the Australian lungfish Neoceratodus forsteri (Krefft 1870)

Australian lungfish Neoceratodus forsteri may be the closest living relative to the first tetrapods and yet little is known about their retinal ganglion cells. This study reveals that lungfish possess a heterogeneous population of ganglion cells distributed in a horizontal streak across the retinal meridian, which is formed early in development and maintained through to adult stages. The number and complement of both ganglion cells and a population of putative amacrine cells within the ganglion cell layer are examined using retrograde labelling from the optic nerve and transmission electron-microscopic analysis of axons within the optic nerve. At least four types of retinal ganglion cells are present and lie predominantly within a thin ganglion cell layer, although two subpopulations are identified, one within the inner plexiform and the other within the inner nuclear layer. A subpopulation of retinal ganglion cells comprising up to 7% or the total population are significantly larger (> 400 mu m(2)) and are characterized as giant or alpha-like cells. Up to 44% of cells within the retinal ganglion cell layer represent a population of presumed amacrine cells. The optic nerve is heavily fasciculated and the proportion of myelinated axons increases with body length from 17% in subadults to 74% in adults. Spatial resolving power, based on ganglion cell spacing, is low (1.6-1.9 cycles deg(-1), n = 2) and does not significantly increase with growth. This represents the first detailed study of retinal ganglion cells in sarcopterygian fish, and reveals that, despite variation amongst animal groups, trends in ganglion cell density distribution and characteristics of cell types were defined early in vertebrate evolution.

Local edge detectors: A substrate for fine spatial vision at low temporal frequencies in rabbit retina

Visual acuity is limited by the size and density of the smallest retinal ganglion cells, which correspond to the midget ganglion cells in primate retina and the beta- ganglion cells in cat retina, both of which have concentric receptive fields that respond at either light- On or light- Off. In contrast, the smallest ganglion cells in the rabbit retina are the local edge detectors ( LEDs), which respond to spot illumination at both light- On and light- Off. However, the LEDs do not predominate in the rabbit retina and the question arises, what role do they play in fine spatial vision? We studied the morphology and physiology of LEDs in the isolated rabbit retina and examined how their response properties are shaped by the excitatory and inhibitory inputs. Although the LEDs comprise only similar to 15% of the ganglion cells, neighboring LEDs are separated by 30 - 40 mu m on the visual streak, which is sufficient to account for the grating acuity of the rabbit. The spatial and temporal receptive- field properties of LEDs are generated by distinct inhibitory mechanisms. The strong inhibitory surround acts presynaptically to suppress both the excitation and the inhibition elicited by center stimulation. The temporal properties, characterized by sluggish onset, sustained firing, and low bandwidth, are mediated by the temporal properties of the bipolar cells and by postsynaptic interactions between the excitatory and inhibitory inputs. We propose that the LEDs signal fine spatial detail during visual fixation, when high temporal frequencies are minimal.

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Expression-based strategies for discovery of genes involved in testis and ovary development

In recent years, strategies for gene identification based on differential gene expression have become increasingly popular, due in part to the development of microarray technology. These strategies are particularly well suited to the identification of genes involved in sex determination and gonadal development, which unlike the development of other organ systems, proceeds along two very different alternative courses, depending on the sex of the embryo. We have used a high-throughput, array-based expression screen to identify several genes expressed sex-specifically in developing mouse gonads. One of these, vanin 1, appears to play a role in mediating migration of mesonephric cells into the male genital ridge. Progress in characterizing other genes arising from the screen is discussed.

Habits and attitudes towards retinoscopy and the relative accuracy of dedicated and combined retinoscopes

The studies presented in this thesis were carried out because of a lack of previous research with respect to (a) the habits and attitudes towards retinoscopy and (b) the relative accuracy of dedicated retinoscopes compared to combined types in which changing the bulb allows use in spot or streak mode. An online British survey received responses from 298 optometrists. Decision tree analyses revealed that optometrists working in multiple practices tended to rely less on retinoscopy than those in the independent sector. Only half of the respondents used dynamic retinoscopy. The majority, however, agreed that retinoscopy was an important test. The University attended also influenced the type of retinoscope used and the use of autorefractors. Combined retinoscopes were used most by the more recently qualified optometrists and few agreed that combined retinoscopes were less accurate. A trial indicated that combined and dedicated retinoscopes were equally accurate. Here, 4 optometrists (2 using spot and 2 using streak retinoscopes) tested one eye of 6 patients using combined and dedicated retinoscopes. This trial also demonstrated the utility of the relatively unknown ’15 degrees of freedom’ rule that exploits replication in factorial ANOVA designs to achieve sufficient statistical power when recruitment is limited. An opportunistic international survey explored the use of retinoscopy by 468 practitioners (134 ophthalmologists, 334 optometrists) attending contact related courses. Decision tree analyses found (a) no differences in the habits of optometrists and ophthalmologists, (b) differences in the reliance on retinoscopy and use of dynamic techniques across the participating countries and (c) some evidence that younger practitioners were using static and dynamic retinoscopy least often. In conclusion, this study has revealed infrequent use of static and dynamic retinoscopy by some optometrists, which may be the only means of determining refractive error and evaluating accommodation in patients with communication difficulties.

The isolation and characterization of tescalcin, a novel gene differentially expressed in the mouse testis during the early stages of gonadal differentiation

Gonadal development is an ideal model to study organogenesis because a variety of developmental processes can be studied during the differentiation of the bipotential primordium into testis or ovary. To better understand this process, Representational Difference Analysis of cDNA was used to identify genes that are differentially expressed in mouse gonads at 13.5 days post-coitus. The analysis led to the identification of three testis specific genes and a sequence that was only expressed in the ovary. The male genes identified: renin, Col9a3, and a novel gene termed tescalcin had patterns of expression that suggested a role in testis determination. ^ Studies of the tescalcin gene revealed that it is organized into eight exons and seven introns. The gene was located at 64 cM in mouse chromosome 5, where it spans approximately 35 Kb. Three mRNA variants resulting from alternative splicing of intron 5 were identified in mouse tissues. Gel mobility shift assays demonstrated that Sp1 and Sp3 from Y-1, msc-1, and MIN-6 cells nuclear extracts bind the GC-boxes within the tescalcin proximal promoter. Bisulfite sequencing analysis of tescalcin CpG island revealed that it is differentially methylated in male and female mouse embryonic gonads, and that hypermethylation of this region represses expression of tescalcin in the β-TC3 cell line. ^ The major tescalcin mRNA encodes a protein with 214 amino acids that contains a consensus EF-hand Ca2+-binding domain and an N-myristoylation motif. The amino acid sequence of tescalcin is highly conserved among various species, and it showed the highest homology with calcineurin B homologous proteins 1 and 2, and calcineurin B. Western blot analysis using antibodies generated against the tescalcin protein confirmed its presence in specific mouse tissues and cell lines. Immunohistochemical analysis of mouse embryos confirmed the pattern of expression of tescalcin mRNA in fetal testis. Using pull-down assays, glyceraidehydes-3-phosphate dehydrogenase was identified as an interacting and potential functional partner of tescalcin. ^ The identification and characterization of tescalcin as a novel embryonic testicular marker will contribute to the elucidation of the genetic pathways involved in testis development and likely to the understanding of pathological conditions such as sex reversal and infertility. ^

(Table 1) Chemical composition of palygorskite and host sediments from Core VITYAZ4599-GC

Palygorskite streak veinlets were studied in the lower part of a sediment core collected at Station VITYAZ4599 in the central (Mid-Indian) basin of the Indian Ocean. It is suggested that this palygorskite is of hydrothermal origin and was formed in pre-Pliocene time.

Going it alone? North Korea’s adaptability as a small power in a changing world

This article uses small states scholarship to map North Korea’s evolution from a post-colonial small state to a system-influencing state due to its nuclear weapons programme. The framework allows for contributions to: (1) The DPRK literature which in some parts has suggested the future collapse of the state, (2) The small states literature that suggests they can only survive if they integrate larger political and/or economic units, (3) The mainstream IR literature and its dominant realist streak that considers great powers and their will as the main drivers in contemporary world politics.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Effects of hypercapnia on aspects of feeding, nutrition, and growth in the edible sea urchin Lytechinus variegatus held in culture

Land-based aquaculture facilities experience occasional hypercapnic conditions due to the accumulation of the metabolic waste product carbon dioxide. Pre-gonadal Lytechinus variegatus (horizontal diameter=20 mm) were exposed to control (608 µatm pCO2, pH 8.1) or hypercapnic conditions (1738 µatm pCO2, pH 7.7) in synthetic seawater for 14 weeks. Sea urchins exposed to hypercapnic conditions exhibited significantly slower growth (reduced dry matter production), primarily due to reduced test production. Higher fecal production rates and lower ash absorption efficiency (%) in individuals exposed to hypercapnic conditions suggest the ability to process or retain dietary carbonates may have been affected. Significant increases in neutral lipid storage in the gut and increased soluble protein storage in the gonads of individuals exposed to hypercapnic conditions suggest alterations in nutrient metabolism and storage. Furthermore, organic production and energy allocation increased in the lantern of those individuals exposed to hypercapnic conditions. These results suggest chronic exposure to hypercapnic conditions alters nutrient allocation to organ systems and functions, leading to changes in somatic and reproductive production.

Pteropod eggs released at high pCO2 lack resilience to ocean acidification

The effects of ocean acidification (OA) on the early recruitment of pteropods in the Scotia Sea, was investigated considering the process of spawning, quality of the spawned eggs and their capacity to develop. Maternal OA stress was induced on female pteropods (Limacina helicina antarctica) through exposure to present day pCO2 conditions and two potential future OA states (750??atm and 1200??atm). The eggs spawned from these females, both before and during their exposure to OA, were incubated themselves in this same range of conditions (embryonic OA stress). Maternal OA stress resulted in eggs with lower carbon content, while embryonic OA stress retarded development. The combination of maternal and embryonic OA stress reduced the percentage of eggs successfully reaching organogenesis by 80%. We propose that OA stress not only affects the somatic tissue of pteropods but also the functioning of their gonads. Corresponding in-situ sampling found that post-larval L. helicina antarctica concentrated around 600?m depth, which is deeper than previously assumed. A deeper distribution makes their exposure to waters undersaturated for aragonite more likely in the near future given that these waters are predicted to shoal from depth over the coming decades.