The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 ( Per2)- dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα ( Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 ( Arnt1), Lhcgr, and connexin ( Cx) 43 ( Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.
The Deleted in Azoospermia-Like (DAZL) protein coded by Dazl gene is a germline-specific RNA-binding protein essential for gametogenesis in vertebrates, and the chicken Dazl gene has also been identified in primordial germ cells (PGCs). However, the temporal and spatial expression of chicken DAZL (cDAZL) and its molecular role in germ cell development remain enigmatic. Here, we investigated the subcellular distribution and expression of cDAZL at the various stages by using a polyclonal antibody raised against its C-terminal region and compared them with those of additional germline-specific proteins chicken vasa homologue (CVH) and chicken dead end homologue (CDH). Western blot analysis for cDAZL revealed a single band in the embryonic gonads and premature chicken testis, whereas no band was detected in the premature chicken ovary. Fluorescent immunohistochemistry revealed that cDAZL was present in the nucleus and cytoplasm of circulating PGCs. Cells positive for cDAZL and CVH coexisted in the embryonic gonads and premature chicken testis, in which they were distributed near the basement membrane of seminiferous tubules. Of interest, cDAZL was not found in the premature chicken ovary, whereas CVH and CDH were present in germ cells. Collectively, three germline-specific proteins are expressed in chicken germ cells, but their patterns of expression are temporally and spatially distinct.
Most of the α-halo carbonyl (AHC) compounds tend to be predicted as mutagenic by structure-activity relationship based on structural category only, because they have an alkyl halide structure as a structural alert of mutagenicity. However, some AHC compounds are not mutagenic. We hypothesized that AHC reacts with DNA by S2 reaction, and the reactivity relates to mutagenicity. As an index of S2 reactivity, we focused on molecular orbitals (MOs), as the direction and position of two molecules in collision are important in the S2 reaction. The MOs suitable for S2 reaction (SN2MOs) were selected by chemical-visual inspection based on the shape of the MO. We used the level gap and the energy gap between SN2MO and the lowest unoccupied molecular orbital as the descriptors of S2 reactivity. As the results, S2 reactivity related to mutagenicity and we were able to predict mutagenicity of 20 AHC compounds with 95.0% concordance. It was suggested that S2 reaction is a reaction mechanism of AHC compounds and DNA in the mutagenic process. The method allows for discrimination among structurally similar compounds by combination with quantitative structure-activity relationships. The combination approach is expected to be useful for the mutagenic assessment of pharmaceutical impurities.
Germ cell-specific genes such as Ddx4, Dnd1, and Dazl play critical roles in the proliferation and survival of germ cells. However, the methylation state of the promoter in mature germ cells is still unknown. Here, we investigated the methylation levels of these genes and the pluripotency marker gene Nanog in chicken sperm as compared with the Alb gene in the liver. CpG islands and/or promoter motifs such as TATA box, GC box and CAAT box were found within the putative promoter regions that we identified. By using the bisulfite reaction, CpG sites in the putative promoters were converted, and they were analyzed by sequencing. The putative promoters of Ddx4, Dnd1, Dazl and Nanog showed very low methylation levels in sperm, but they were highly methylated in the liver. Conversely, the Alb gene promoter was highly methylated in sperm and hypomethylated in the liver. However, no transcripts of Ddx4, Dnd1, Dazl and Nanog were detected in sperm or the liver. Also, no transcripts of Dnmt1 and Dnmt3a were detected in sperm. Our present results may indicate that these germ cell-specific genes and the pluripotency marker gene are ready to express any time after fertilization. Our findings showing that low methylation and selective DNA methylation of specific genes are present in chicken sperm contribute to our understanding of fertilization and embryogenesis of birds.
Introduction: Several studies published mainly from pioneers and early adopters have documented the evolution of minimally-invasive hepatectomy(MIH). However, questions remain if these reported experiences are applicable and reproducible today. This study examines the changing trends, safety and outcomes associated with the adoption of MIH Method: Retrospective review of 500 consecutive patients who underwent MIH between 2006-2018 of which 460 cases (92%) were performed since 2012. To determine the evolution of MIH, the study population was stratified into 5 equal groups of 100 patients. Analyses was also performed of predictive factors and outcomes of open conversion. Result: Five hundred patients underwent MIH of which 479 (97.8%) were totally laparoscopic/robotic. 118 (23.6%) patients underwent major hepatectomy and 199 (39.9%) had resection of tumors located in the posterosuperior segments. 32 patients (6.4%) had previous liver resections. There were 45 (9.0%) open conversions.Comparison across the 5 groups demonstrated that patients were older, had higher ASA score, had increased frequency of previous abdominal surgery and repeat liver resections. There was also an increase in the proportion of patients who underwent totally laparoscopic/robotic surgery, major liver resection, resection of 3 segments and multiple resections. Comparison of outcomes demonstrated that there was a significant decrease in open conversion rate, longer operation time and increased use of Pringles maneuver. Presence of cirrhosis and institution experience (1 st 100 cases) were independent predictors of open conversion. Patients who required open conversion had significantly increased operation time, blood loss, blood transfusion rate, morbidity and mortality. Conclusion:The case volume of MIH performed increased rapidly at our institution over time. Although the indications of MIH expanded to include higher risk patients and more complex hepatectomies, there was a decrease in open conversion rate and no change in other perioperative outcomes.
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