No abstract

Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24 and 36 h) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n= 4-8/timepoint), with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1 and HSD11B2), steroidogenic acute regulatory protein (StAR) and gonadotrophin receptors [LH/choriogonadotrophin receptor (LHCGR), FSH receptor (FSHR)] as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for epidermal-growth-factor-like factors (amphiregulin, epiregulin) and processes [hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6)] implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g. CYP17A), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell and surface epithelium (HSD11B)-related genes in the rodent/primate pre-ovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response and angiogenesis), and for identifying novel gene products controlling mammalian fertility.

During the formation of the enteric nervous system (ENS) of the moth Manduca sexta, identified populations of neurons and glial cells participate in precisely timed waves of migration. The cell adhesion receptor fasciclin II is expressed in the developing ENS and is required for normal migration. Previously, we identified two isoforms of Manduca fasciclin II (MFas II), a glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) and a transmembrane isoform (TM-MFas II). Using RNA and antibody probes, we found that these two isoforms were expressed in cell type-specific patterns: GPI-MFas II was expressed by glial cells and newly generated neurons, while TM-MFas II was confined to differentiating neurons. The expression of each isoform also corresponded to the motile state of the different cell types: GPI-MFas II was detected on tightly adherent or slowly spreading cells, while TM-MFas II was expressed by actively migrating neurons and was localized to their most motile regions. Manipulations of each isoform in embryo culture showed that they played distinct roles: whereas GPI-MFas II acted strictly as an adhesion molecule, TM-MFas II promoted the motility of the EP cells as well as maintaining fasciculation with their pathways. These results indicate that precisely regulated patterns of isoform expression govern the functions of fasciclin II within the developing nervous system.

Neuronal differentiation requires a coordinated intracellular response to diverse extracellular stimuli, but the role of specific signaling mechanisms in regulating this process is still poorly understood. Soluble guanylate cyclases (sGCs), which can be stimulated by diffusible free radical gasses such as nitric oxide (NO) and carbon monoxide (CO) to produce the intracellular messenger cGMP, have recently been found to be expressed within a variety of embryonic neurons and implicated in the control of both neuronal motility and differentiation. Using the enteric nervous system (ENS) of the moth, Manduca sexta, we examined the role of NO and NO-sensitive sGCs during the migration and differentiation of an identified set of migratory neurons (the EP cells). Shortly after the onset of their migration, a subset of EP cells began to express NO-sensitive sGC activity (visualized with an anti-cGMP antiserum). Unlike many neurons in the central nervous system, the expression of sGC activity in the EP cells was not transient but persisted throughout subsequent periods of axon elongation and terminal branch formation on the gut musculature. In contrast, nitric oxide synthase activity (visualized using NADPH-diaphorase histochemistry) was undetectable in the vicinity of the EP cells until the period of synapse formation. Manipulations designed to alter sGC and NOS activity in an in vivo embryonic culture preparation had no discernible effect on either the migration or axonal outgrowth of the EP cells. In contrast, inhibition of both of these enzymes resulted in a significant reduction in terminal synaptic branch formation within the postmigratory neurons. These results indicate that while NO-sensitive sGC activity is expressed precociously within the EP cells during their initial migratory dispersal, a role for this signaling pathway can only be demonstrated well after migration is complete, coincident with the formation of mature synaptic connections.

BackgroundThe consequences of defective homologous recombination (HR) are not understood in sporadic ovarian cancer, nor have the potential role of HR proteins other than BRCA1 and BRCA2 been clearly defined. However, it is clear that defects in HR and other DNA repair pathways are important to the effectiveness of current therapies. We hypothesize that a subset of sporadic ovarian carcinomas may harbor anomalies in HR pathways, and that a BRCAness profile (defects in HR or other DNA repair pathways) could influence response rate and survival after treatment with platinum drugs. Clinical availability of a BRCAness profile in patients and/or tumors should improve treatment outcomes.ObjectiveTo define the BRCAness profile of sporadic ovarian carcinoma and determine whether BRCA1, PARP, FANCD2, PTEN, H2AX, ATM, and P53 protein expression correlates with response to treatment, disease recurrence, and recurrence-free survival.Materials and MethodsProtein microarray analysis of ovarian cancer tissue was used to determine protein expression levels for defined DNA repair proteins. Correlation with clinical and pathologic parameters in 186 patients with advanced stage III–IV and grade 3 ovarian cancer was analyzed using Chi square, Kaplan-Meier method, Cox proportional hazard model, and cumulative incidence function.ResultsHigh PARP, FANCD2 and BRCA1 expressions were significantly correlated with each other; however, elevated p53 expression was associated only with high PARP and FANCD2. Of all patients, 9% recurred within the first year. Among early recurring patients, 41% had high levels of PARP, FANCD2 and P53, compared to 19.5% of patients without early recurrence (p = 0.04). Women with high levels of PARP, FANCD2 and/or P53 had first year cumulative cancer incidence of 17% compared with 7% for the other groups (P = 0.03).ConclusionsPatients with concomitantly high levels of PARP, FANCD2 and P53 protein expression are at increased risk of early ovarian cancer recurrence and platinum resistance.