The effect of subluteal levels of exogenous progesterone on follicular dynamics and endocrine patterns during early luteal phase of the ewe

Nineteen Corriedale ewes were treated with an im dose of a PGF2alpha during the luteal phase to synchronize estrus. After ovulation had been detected by using ultrasonography (Day 0); the ewes were randomly assigned to 2 different groups. In 11 ewes a CIDR, which had previously been used for 10 d, was inserted on the fourth day after ovulation. The ewes then received a dose of PGF2alpha on Day 5 to induce luteolysis. The CIDR remained in place until the end of the experiment (Day 9). Control ewes (n = 8) received no treatment. Blood samples were taken daily for estradiol, progesterone and FSH determinations. In the untreated ewes, 2 follicular waves were detected in all of the animals throughout the monitoring period, with a mean wave interval of 4.5 d. The total number of follicles which were > or =2 mm decreased from Day 0 to Day 4 (8.8+/-1.0 to 5.3+/-0.6; P< or =0.05) and then increased at Day 7 (7.5+/-0.9; P< or =0.05). The growth profiles of both the largest and the second largest follicles of Wave 1 showed significant divergence, while no divergence was observed in Wave 2. Serum estradiol concentrations decreased significantly from the day before to the day of ovulation and then increased again during the growing phase of the largest follicle of Wave 1. Concentrations of FSH were high on the day of emergence of both waves, but while a significant decline was observed after emergence in Wave 1, the levels remained high in Wave 2. In 8 of the 11 treated ewes, the largest follicle of Wave 1 was still present on the ninth day after ovulation (persistent follicle). In the other 3 ewes, the largest follicle of Wave 1 was already regressing on the day that the treatment was administered, and the largest follicle that was present on Day 9 originated from Wave 2 (nonpersistent follicle). In persistent follicle ewes, the largest follicle of Wave 1 prolonged its lifespan significantly, attaining the maximum diameter (Day 8.1+/-0.8) later than in untreated (Day 3.0+/-0.4) and nonpersisted follicle ewes (Day 2.0+/-0.6). The total number of follicles decreased in persistent follicle ewes between Day 0 and Day 4 (7.9+/-1.5 to 4.5+/-0.5, respectively; P< or =0.05) and remained low until the end of the experiment. Progesterone concentrations (nmol/L) between Days 6 and 9 were significantly different between untreated and persistent follicle ewes (12.8+/-1.0 vs. 9.4+/-1.0, P< or =0.02). The present study confirms that the largest follicle of Wave 1 is dominant in the ewe and that subluteal progesterone concentrations can prolong its lifespan and extend this dominance.     

A Raf-1 mutant that dissociates MEK/extracellular signal-regulated kinase activation from malignant transformation and differentiation but not proliferation

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.     

Profiling oligosaccharidurias by electrospray tandem mass spectrometry: quantifying reducing oligosaccharides

A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Skeletal Muscle Insulin Resistance Associated with Cholesterol-Induced Activation of Macrophages Is Prevented by High Density Lipoprotein

Background

Emerging evidence suggests that high density lipoprotein (HDL) may modulate glucose metabolism through multiple mechanisms including pancreatic insulin secretion as well as insulin-independent glucose uptake into muscle. We hypothesized that HDL may also increase skeletal muscle insulin sensitivity via cholesterol removal and anti-inflammatory actions in macrophages associated with excess adiposity and ectopic lipid deposition.

Methods

Human primary and THP-1 macrophages were treated with vehicle (PBS) or acetylated low density lipoprotein (acLDL) with or without HDL for 18 hours. Treatments were then removed, and macrophages were incubated with fresh media for 4 hours. This conditioned media was then applied to primary human skeletal myotubes derived from vastus lateralis biopsies taken from patients with type 2 diabetes to examine insulin-stimulated glucose uptake.

Results

Conditioned media from acLDL-treated primary and THP-1 macrophages reduced insulin-stimulated glucose uptake in primary human skeletal myotubes compared with vehicle (primary macrophages, 168±21% of basal uptake to 104±19%; THP-1 macrophages, 142±8% of basal uptake to 108±6%; P<0.05). This was restored by co-treatment of macrophages with HDL. While acLDL increased total intracellular cholesterol content, phosphorylation of c-jun N-terminal kinase and secretion of pro- and anti-inflammatory cytokines from macrophages, none were altered by co-incubation with HDL. Insulin-stimulated Akt phosphorylation in human skeletal myotubes exposed to conditioned media was unaltered by either treatment condition.

Conclusion

Inhibition of insulin-stimulated glucose uptake in primary human skeletal myotubes by conditioned media from macrophages pre-incubated with acLDL was restored by co-treatment with HDL. However, these actions were not linked to modulation of common pro- or anti-inflammatory mediators or insulin signaling via Akt.     

Uterine estrogen receptor alpha and progesterone receptor during the follicular and luteal phase in llamas

Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were characterized in different endometrial cell types as luminal and glandular epithelium and stroma during the follicular (FP) and the luteal phase (LP) in llamas. Animals were examined daily by transrectal ultrasonography for the determination of the presence of an ovulatory follicle and ovulation was immediately induced by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left uterine horn on Day 0 (FP) and 9 days after the GnRH injection (Day 9, LP). Blood samples were collected on these days for estradiol 17beta and progesterone determination by RIA. An immunohistochemical technique was used to visualize ERalpha and PR immunostaining which was then analyzed by two independent observers. Total positive area and average staining for ERalpha were affected by the phase of the ovarian activity: in the three cell types there was more positive area and intense staining during the FP than during the LP. Similar findings were observed for PR, more positive stained areas were found during the FP than during the LP in the epithelia. In addition, the three cell types had more intense staining during the FP than during the LP. An effect of the cell type for ERalpha and PR was observed; epithelia (luminal and glandular) had more positive stained areas and greater intensity than stromal cells. In conclusion, the results of the present study suggest that in llamas, like in other ruminants, estradiol has a stimulatory effect while progesterone downregulates the ERalpha and PR and that the receptor is cell type specific.