Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes

Background: At later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small ( 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets).Results: Principal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues.Conclusions: Our data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells. © 2014 Hatzirodos et al.; licensee BioMed Central Ltd.Nicholas Hatzirodos, Helen F Irving-Rodgers, Katja Hummitzsch, Margaret L Harland, Stephanie E Morris and Raymond J Rodger

Transcriptome profiling of granulosa cells from bovine ovarian follicles during atresia

Background: The major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm). Results: Principal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-beta and tumour necrosis factor-alpha pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-beta signalling and were up regulated. Conclusions: Small healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.Nicholas Hatzirodos, Katja Hummitzsch, Helen F Irving-Rodgers, Margaret L Harland, Stephanie E Morris and Raymond J Rodger

A molecular perspective on the limits of life: Enzymes under pressure

From a purely operational standpoint, the existence of microbes that can grow under extreme conditions, or "extremophiles", leads to the question of how the molecules making up these microbes can maintain both their structure and function. While microbes that live under extremes of temperature have been heavily studied, those that live under extremes of pressure have been neglected, in part due to the difficulty of collecting samples and performing experiments under the ambient conditions of the microbe. However, thermodynamic arguments imply that the effects of pressure might lead to different organismal solutions than from the effects of temperature. Observationally, some of these solutions might be in the condensed matter properties of the intracellular milieu in addition to genetic modifications of the macromolecules or repair mechanisms for the macromolecules. Here, the effects of pressure on enzymes, which are proteins essential for the growth and reproduction of an organism, and some adaptations against these effects are reviewed and amplified by the results from molecular dynamics simulations. The aim is to provide biological background for soft matter studies of these systems under pressure.Comment: 16 pages, 8 figure

Information content of ozone retrieval algorithms

The algorithms are characterized that were used for production processing by the major suppliers of ozone data to show quantitatively: how the retrieved profile is related to the actual profile (This characterizes the altitude range and vertical resolution of the data); the nature of systematic errors in the retrieved profiles, including their vertical structure and relation to uncertain instrumental parameters; how trends in the real ozone are reflected in trends in the retrieved ozone profile; and how trends in other quantities (both instrumental and atmospheric) might appear as trends in the ozone profile. No serious deficiencies were found in the algorithms used in generating the major available ozone data sets. As the measurements are all indirect in someway, and the retrieved profiles have different characteristics, data from different instruments are not directly comparable

Transcriptome comparisons identify new cell markers for theca interna and granulosa cells from small and large antral ovarian follicles

In studies using isolated ovarian granulosa and thecal cells it is important to assess the degree of cross contamination. Marker genes commonly used for granulosa cells include FSHR, CYP19A1 and AMH while CYP17A1 and INSL3 are used for thecal cells. To increase the number of marker genes available we compared expression microarray data from isolated theca interna with that from granulosa cells of bovine small (n = 10 for both theca and granulosa cells; 3-5 mm) and large (n = 4 for both theca and granulosa cells, > 9 mm) antral follicles. Validation was conducted by qRT-PCR analyses. Known markers such as CYP19A1, FSHR and NR5A2 and another 11 genes (LOC404103, MGARP, GLDC, CHST8, CSN2, GPX3, SLC35G1, CA8, CLGN, FAM78A, SLC16A3) were common to the lists of the 50 most up regulated genes in granulosa cells from both follicle sizes. The expression in theca interna was more consistent than in granulosa cells between the two follicle sizes. Many genes up regulated in theca interna were common to both sizes of follicles (MGP, DCN, ASPN, ALDH1A1, COL1A2, FN1, COL3A1, OGN, APOD, COL5A2, IGF2, NID1, LHFP, ACTA2, DUSP12, ACTG2, SPARCL1, FILIP1L, EGFLAM, ADAMDEC1, HPGD, COL12A1, FBLN5, RAMP2, COL15A1, PLK2, COL6A3, LOXL1, RARRES1, FLI1, LAMA2). Many of these were stromal extracellular matrix genes. MGARP, GLDC, CHST8, GPX3 were identified as new potential markers for granulosa cells, while FBLN5, OGN, RAMP2 were significantly elevated in the theca interna.Nicholas Hatzirodos, Katja Hummitzsch, Helen F. Irving-Rodgers, Raymond J. Rodger

Transcriptome profiling of the theca interna from bovine ovarian follicles during atresia

The theca interna is a specialized stromal layer that envelops each growing ovarian follicle. It contains capillaries, fibroblasts, immune cells and the steroidogenic cells that synthesize androgens for conversion to estradiol by the neighboring granulosa cells. During reproductive life only a small number of follicles will grow to a sufficient size to ovulate, whereas the majority of follicles will undergo regression/atresia and phagocytosis by macrophages. To identify genes which are differentially regulated in the theca interna during follicular atresia, we undertook transcriptome profiling of the theca interna from healthy (n = 10) and antral atretic (n = 5) bovine follicles at early antral stages (<5 mm). Principal Component Analyses and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. A total of 543 probe sets were differentially expressed between the atretic and healthy theca interna. Further analyses of these genes by Ingenuity Pathway Analysis and Gene Ontology Enrichment Analysis Toolkit software found most of the genes being expressed were related to cytokines, hormones and receptors as well as the cell cycle and DNA replication. Cell cycle genes which encode components of the replicating chromosome complex and mitotic spindle were down-regulated in atretic theca interna, whereas stress response and inflammation-related genes such as TP53, IKBKB and TGFB1 were up-regulated. In addition to cell cycle regulators, upstream regulators that were predicted to be inhibited included Retinoblastoma 1, E2 transcription factor 1, and hepatocyte growth factor. Our study suggests that during antral atresia of small follicles in the theca interna, arrest of cell cycle and DNA replication occurs rather than up- regulation of apoptosis-associated genes as occurs in granulosa cells.Nicholas Hatzirodos, Helen F. Irving-Rodgers, Katja Hummitzsch, Raymond J. Rodger

Probable Gravitational Microlensing towards the Galatic Bulge

The MACHO project carries out regular photometric monitoring of millions of stars in the Magellanic Clouds and Galactic Bulge, to search for very rare gravitational microlensing events due to compact objects in the galactic halo and disk. A preliminary analysis of one field in the Galactic Bulge, containing {$\sim430,000$} stars observed for 190 days, reveals four stars which show clear evidence for brightenings which are time-symmetric, achromatic in our two passbands, and have shapes consistent with gravitational microlensing. This is significantly higher than the $\sim 1$ event expected from microlensing by known stars in the disk. If all four events are due to microlensing, a 95\% confidence lower limit on the optical depth towards our bulge field is $1.3 \times 10^{-6}$, and a ``best fit" value is $\tau \approx 1.6 \times 10^{-6}/\epsilon$,where $\epsilon$ is the detection efficiency of the experiment, and $\epsilon < 0.4$. If the true optical depth is close to the ``best fit" value, possible explanations include a ``maximal" disk which accounts for most of the galactic circular velocity at the solar radius, a halo which is centrally concentrated, or bulge-bulge microlensing.Comment: submitted to Astrophysical Journal Letters, 10 pages text as uuencoded compressed PostScript, 5 figures and paper also available via anonymous ftp from merlin.anu.edu.au in /pub/kcf/mach

Nonlinear models of the bump cepheid HV 905 and the distance modulus to the large magellanic cloud

Nonlinear pulsation models have been used to simulate the light curve of the LMC bump Cepheid HV 905. In order to reproduce the light curve accurately, tight constraints on the input parameters M, L, and T-eff are required. The results, combined with accurate existing V and I photometry, yield an LMC distance modulus of 18.51 +/- 0.05, and they show that the luminosity of HV 905 is much higher than expected from the mass-luminosity relation of stellar evolution theory. If we assume that the pulsation models are accurate, this suggests that there is a larger amount of convective core overshoot during the main-sequence evolution of stars with M similar to 5 M. than is usually assumed

Correlated Microscopic Observations of Arterial Responses to Intravascular Stenting

Percutaneous catheter implantation of intravascular stent prostheses has emerged as a novel clinical adjunct to balloon angioplasty in the treatment of obstructive atherosclerotic vascular disease. We have examined the cellular and subcellular responses to stenting in the coronary arteries of the dog and pig (both normal and atherosclerotic), and in the iliac arteries and aorta of the atherosclerotic rabbit, using scanning electron, transmission electron, and light microscopies. Stenting in these models resulted in a thrombotic reaction ranging from mild to severe, depending on species and antithrombotic therapy. Subsequent organization of thrombotic material with hyperplasia of smooth muscle and inflammatory cells, luminal recovering with endothelial or pseudoendothelial cells, and atrophy of the tunica media led to incorporation of the prosthesis into the arterial wall. Endothelial or pseudoendothelial cells were observed adherent to the prosthesis as early as one day after placement, and regeneration of a confluent periluminal cell layer occurred within 2 to 4 weeks. Persistent ultrastructural abnormalities of the periluminal cell layer were seen as late as 2 years after stenting, but the intimal hyperplastic response appeared limited