Enhanced method to select human oogonial stem cells for fertility research.

Alternative methods to obtain mature oocytes are still needed for women with premature ovarian failure (POF). Oogonial stem cells (OSCs), found in adult ovaries, have provided insight into potential paths to treating infertility. Previously, the DDX4 antibody marker alone was utilized to isolate OSCs; however, extensive debate over its location in OSCs versus resulting oocytes (transmembrane or intracytoplasmic) has raised doubt about the identity of these cells. Separate groups, however, have efficiently isolated OSCs using another antibody marker Ifitm3 which is consistently recognized to be transmembrane in location. We hypothesized that by using anti-DDX4 and anti-IFITM3 antibodies, in combination, with MACS, we would improve the yield of isolated OSCs versus using anti-DDX4 antibodies alone. Our study supports earlier findings of OSCs in ovaries during the entire female lifespan: from reproductive age through post-menopausal age. MACS sorting ovarian cells using a the two-marker combination yielded a ~ twofold higher percentage of OSCs from a given mass of ovarian tissue compared to existing single marker methods while minimizing the debate surrounding germline marker selection. During in vitro culture, isolated cells retained the germline phenotype expression of DDX4 and IFITM3 as confirmed by gene expression analysis, demonstrated characteristic germline stem cell self-assembly into embryoid bodies, and formed > 40 µm "oocyte-like" structures that expressed the early oocyte markers GDF9, DAZL, and ZP1. This enhanced and novel method is clinically significant as it could be utilized in the future to more efficiently produce mature, secondary oocytes, for use with IVF/ICSI to treat POF patients.

Microfluidic Systems for Assisted Reproductive Technologies: Advantages and Potential Applications.

Microfluidic technologies have emerged as a powerful tool that can closely replicate the in-vivo physiological conditions of organ systems. Assisted reproductive technology (ART), while being able to achieve successful outcomes, still faces challenges related to technical error, efficiency, cost, and monitoring/assessment. In this review, we provide a brief overview of the uses of microfluidic devices in the culture, maintenance and study of ovarian follicle development for experimental and therapeutic applications. We discuss existing microfluidic platforms for oocyte and sperm selection and maintenance, facilitation of fertilization by in-vitro fertilization/intracytoplastimc sperm injection, and monitoring, selection and maintenance of resulting embryos. Furthermore, we discuss the possibility of future integration of these technologies onto a single platform and the limitations facing the development of these systems. In spite of these challenges, we envision that microfluidic systems will likely evolve and inevitably revolutionize both fundamental, reproductive physiology/toxicology research as well as clinically applicable ART.

Myeloid atg5 deletion impairs n-3 PUFA-mediated atheroprotection.

BACKGROUND AND AIMS: Dietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. METHODS: We generated myeloid-specific autophagy-deficient and control mice in the Ldlr-/- background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr-/- recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. RESULTS: Compared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr-/- mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr-/- mice. CONCLUSIONS: Deficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.

Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy.

Dietary PUFAs reduce atherosclerosis and macrophage inflammation, but how nucleotide-binding oligomerization domain leucine-rich repeat-containing receptor protein (NLRP3) inflammasome activation and autophagy influence PUFA-mediated atheroprotection is poorly understood. We fed Ldlr-/- mice diets containing 10% (calories) palm oil (PO) and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, fish oil (FO), echium oil (EO, containing 18:4 n-3), or borage oil (BO, containing 18:3 n-6). Inflammasome activation, autophagic flux, and mitochondrial function were measured in peritoneal macrophages, blood monocytes, or liver from diet-fed mice. Compared with PO, dietary PUFAs (FO, EO, or BO) markedly inhibited inflammasome activation, shown by 1) less macrophage IL-1ß secretion and caspase-1 cleavage in response to NLRP3 inflammasome activators, 2) less IL-1ß secretion and caspase-1 cleavage from liver or hepatocytes in response to lipopolysaccharide (LPS), and 3) attenuated caspase-1 activity in blood monocytes. Furthermore, PUFA-enriched diets increased LC3-II expression in macrophage, aorta, and liver samples and reduced numbers of dysfunctional mitochondria in macrophages in response to LPS and palmitate, suggesting enhanced autophagic activation. Dietary PUFAs did not attenuate NLRP3 inflammasome activation in atg5-deficient macrophages, indicating that autophagic activation is critical for the PUFA-mediated inflammasome inactivation. In conclusion, dietary PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.

Platinum-acridinylthiourea conjugates show cell line-specific cytotoxic enhancement in H460 lung carcinoma cells compared to cisplatin.

Recently, we reported a new class of DNA-targeted hybrid platinum-acridine agents. The parent intercalator, ACRAMTU, a 9-aminoacridine derivative, intercalates into the minor groove of DNA, causing the corresponding prototypical conjugate, PT-ACRAMTU (type I/n=2), to form DNA adducts dissimilar to traditional platinum drugs. Both these agents show cytotoxic activity in leukemic and ovarian cancer cells. Following the use of clonogenic survival assays, we report on the cytotoxic effects of ACRAMTU, PT-ACRAMTU, and three PT-ACRAMTU derivatives, on additional cell lines including colon (RKO), lung (H460), and cisplatin-sensitive (A2780) and cisplatin-resistant (A2780/CP) ovarian cells. While a dose-dependent effect was observed with both ACRAMTU and PT-ACRAMTU, an enhanced cytotoxic effect was seen with PT-ACRAMTU in all cell lines. PT-ACRAMTU appeared to have a similar IC50 value to cisplatin except in H460 lung cancer cells in which PT-ACRAMTU had a twofold lower IC50 value. PT-ACRAMTU appeared to act in a time-dependent manner. In H460 cells the IC50 value of PT-ACRAMTU was 235-fold higher following a 1-h incubation than following a 24-h incubation (0.27 microM), while following an 8-h incubation the IC50 value was 0.41 microM. Three derivatives of PT-ACRAMTU were also tested. A tetraalkylated derivative, type II/n=2, generated the highest IC50 values in all cell lines, while the trialkylated derivative, type III/n=2, generated IC50 values similar to its isomer, PT-ACRAMTU. PT-ACRAMTU with an added CH2 group in the thiourea linker (type I/n=3) showed IC50 values similar to the type I/n=2 prototype in H460 lung cells. An apoptotic response to PT-ACRAMTU appeared to be generated in H460 cells as evidenced by DNA laddering. These results suggest that type I/n=2 and type I/n=3 may be promising agents for the treatment of lung cancer and should be pursued in animal models.