Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Involvement of nuclear factor kappa B in the regulation of rat luteal function: potential roles as survival factor and inhibitor of 20alpha-hydroxysteroid dehydrogenase.

Nuclear factor kappa B (NFkappaB) is an important intracellular conveyor of extracellular signals and modulates a number of gene responses. Due to the potential significance of NFkappaB in regulating ovarian gene expression, we examined in the rat: (i) whether NFkappaB is activated and developmentally regulated in the corpus luteum (CL) throughout pregnancy; (ii) the proteins forming the NFkappaB complex in luteal cells; and (iii) the role of this transcription factor in luteal function. Western analysis and immunohistochemistry revealed that p65 and p50 were highly expressed throughout pregnancy and were located in both the nucleus and cytoplasm of luteal cells. In addition, because NFkappaB is maintained in the cytoplasm bound to IkappaB, whose phosphorylation allows NFkappaB translocation to the nucleus, we studied the developmental expression of phosphorylated and nonphosphorylated forms of IkappaBalpha. Western analysis revealed that IkappaBalpha was present and phosphorylated throughout pregnancy in the CL whereas by protein/DNA array and electromobility shift assays we found that luteal nuclear extracts bind to an NFkappaB consensus sequence, and that the binding activity decreased along pregnancy. The specific binding was supershifted only by an anti-p65 antibody and not by antibodies against p50, p52, cRel, or RelB. Using day 4 postpartum ovaries, we found higher NFkappaB binding activity in the newly formed CL than in old CL of pregnancy. Furthermore, NFkappaB DNA binding activity was enhanced by prolactin in luteinized granulosa cells. In our first functional study, blockade of NFkappaB/p65 binding to DNA with the sesquiterpene lactone helenalin in luteinized granulosa cells correlated with induction of cell death in a dose-dependent manner. In a second functional study, overexpression of NFkappaB/p65 in luteal cells resulted in inhibition of 20alpha-hydroxysteroid dehydrogenase (20alphaHSD) promoter activity as well as endogenous 20alphaHSD mRNA expression. In summary, we have shown that: (i) NFkappaB is expressed within the CL, primary luteinized granulosa cells, and a rat luteal cell line; (ii) NFkappaB activation within the CL is developmentally regulated in pregnancy, depends on the age of the gland, and can be upregulated by prolactin; (iii) inhibition of NFkappaB/p65 binding to an NFkappaB DNA consensus sequence correlates with induction of cell death in ovarian luteinized granulosa cells; and (iv) overexpression of NFkappaB in luteal cells inhibits 20alphaHSD gene expression. The results further support a role for NFkappaB as a survival factor in the CL.

Apoptosis induced by antigestagen RU486 in rat corpus luteum of pregnancy.

Administration of RU486 to late pregnant rats results in preterm delivery 24 h after treatment and the induction of a luteolytic process after labor. We investigated whether functional changes occurring within the corpora lutea after RU486 treatment were associated with morphologic features of apoptotic cell death. Rats on d 18 of pregnancy were treated with RU486 (5 mg/kg) at 10:00 am and killed 72 h after. We studied the number of apoptotic cells in paraffin sections of the corpora lutea by routine hematoxylin and eosin (H&E) staining, and by in situ 3' end labeling (TdT-mediated dUTP nick-end labeling [TUNEL]). The corpora lutea were also processed for electron microscopy to study ultrastructural changes after RU486 treatment. The number of cells showing apoptotic nuclei in H&E-stained sections was higher in RU486-treated animals than in controls (vehicle-treated rats). The quantification of the number of apoptotic nuclei within the corpora lutea performed by TUNEL confirmed the higher number of apoptotic nuclei in animals receiving the antigestagen compared with controls. Ultrastructurally, the luteal cells undergoing apoptosis presented a highly deteriorated cytoplasmic organization The nuclei, in an initial step of regression, displayed condensation of the chromatin, a prominent nucleolus, and a perinuclear space. In an advanced step of degeneration, the nuclei showed evidence of large irregular aggregates of condensed chromatin. Prostaglandin F2,alpha(PGF2alpha), which mediates the luteolytic action of RU486, mimicked the effect of the antigestagen on the induction of apoptosis when administered to rats on d 18 of pregnancy (100 microg at 9:00 am and 1:00 pm), which were killed 72 after the last injection. In conclusion, the present results indicate that functional luteolysis in rats is associated with structural luteal regression with the morphologic features of apoptotic cell death, as demonstrated by studying the luteolytic process induced by the administration of the antigestagen RU486.