Effects of Poly (ADP-ribose) Polymerase Inhibition on DNA Integrity and Gene Expression in Ovarian Follicular Cells in Mice with Endotoxemia

Background: A mouse model of lipopolysaccharide (LPS)-induced inflammation was used to investigate the effect of pharmacological inhibition of nuclear enzyme PARP-1 on oocyte maturation, apoptotic and necrotic death, as well as DNA integrity of follicular cells. Also, the relative expression of cumulus genes (HAS2, COX2, and GREM1) associated with oocyte developmental competence was assessed. Methods: Mice were treated with the PARP-1 inhibitor, 4-HQN, one hour before LPS administration. After 24 h, oocyte in vitro maturation was detected. Granulosa cell DNA damage was determined by the alkaline comet assay. Live, necrotic and apoptotic cells were identified using double vital staining by fluorescent dyes, Hoechst 33342 and propidium iodide. The expression levels of cumulus genes were assessed using reverse transcriptase PCR. Results: The administration of 4-HQN to LPS-treated mice ameliorated oocyte meiotic maturation and exerted a significant cytoprotective effect. 4-HQN attenuated LPS-induced DNA damage and favored cell survival by decreasing necrosis and apoptosis in granulosa cells. Exposure to 4-HQN increased mRNA expression levels for HAS2, COX2, and GREM1 in cumulus cells. Conclusion: The obtained results indicate the involvement of PARP-1 in the pathogenesis of ovarian dysfunction caused by LPS. We suppose that this enzyme can be an attractive target for the therapy of inflammatory disorders in ovary. The protective action of PARP-1 inhibition could at least partly be associated with the reduction of necrotic death of follicular cells and also in other cells. However, the detailed mechanisms of the favorable effect of PARP inhibitors on endotoxin-induced ovarian disorders need to be further explored.

Changes in DNA integrity and gene expression in ovarian follicular cells of lipopolysaccharide-treated female mice.

BACKGROUND: Lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, can impair female reproductive function. However, there is a little information about genotoxic stress in ovarian follicular cells as well as about the changes in oocyte developmental potential under endotoxemia. So the aim of our study was to investigate in vitro oocyte maturation, the DNA damage and expression of some developmental competence-related genes in follicular cells of mice treated with LPS. METHODS: LPS (3mg/kg) was intraperitoneally injected into the mice for 24h, and in vitro maturation of mouse oocyte was determined. The expression levels of genes in cumulus cells were detected by reverse transcriptase polymerase chain reaction. DNA damage in granulosa cells was assessed by the alkaline comet assay. RESULTS: LPS injection caused an impairment of oocyte maturation in vitro: the percentage of oocytes reaching metaphase I and metaphase II decreased markedly compared to vehicle control mice. At the same time we observed strong DNA damage in granulosa cells of LPS-treated animals. The endotoxemia resulted in significantly reduced mRNA expression levels for hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX2) and Gremlin-1 (GREM1) genes compared with control. CONCLUSIONS: Our results obtained in a mouse model of endotoxin-induced female reproductive dysfunction suggest that LPS may affect oocyte quality through the induction of DNA damage and decreasing the cumulus expression of genes associated with cumulus expansion and oocyte maturation, such as HAS2, COX2 and GREM1.

Functional effects of antibodies against non-neuronal nicotinic acetylcholine receptors.

Non-neuronal nicotinic acetylcholine receptors (nAChRs) are expressed in the spleen and regulate B lymphocyte propagation and activation. The aim of the present study was to investigate the cellular and physiological effects of antibodies against alpha4(1-209) and alpha7(1-208) nAChR extracellular domains. The antibodies, added in vitro, produced in vivo or injected, specifically bound mouse spleen B lymphocytes. Immunization with nAChR extracellular domains resulted in connective tissue overgrowth and infiltration of segmented neutrophils in the spleen, as well as in decreased body weight compared to mice immunized with BSA. In spite of certain cross-reactivity of alpha4(1-209)- and alpha7(1-208)-specific antibodies, all observed effects were more pronounced upon immunization with alpha7 extracellular domain. Spleens of mice injected with alpha7(1-208)-specific antibody contained decreased numbers of Annexin V-positive B lymphocytes compared to mice injected with non-specific IgG. It is concluded that alpha7 nAChRs are involved in regulating the lymphocyte survival, neutrophil migration, connective tissue overgrowth and body weight accumulation. The antibody binding triggers alpha7 nAChR signaling supporting the idea of non-channel mode of nAChR functioning in B lymphocytes.