Successful Maturation Research Articles

Somatic embryogenesis of Pinus rigida × P. taeda and the relationship between the initiation of embryogenic tissue and zygotic embryo development

The pitch-loblolly pine hybrid (Pinus rigida × P. taeda) has useful characteristics of the parents, but its exploitation is hindered by restrictions of conventional breeding and propagation methods. This study was undertaken to establish an effective in vitro system for propagating pitch-loblolly hybrid pine through somatic embryogenesis and to unravel the relationship between the efficiency of embryogenic tissue initiation and zygotic embryo development. To initiate embryogenic tissue, megagametophytes of developing seeds were used as explants. Seeds were collected weekly, examined, and tested during June and July 2004. The medium and seed collection date were the most important factors for the successful somatic embryogenesis of P. rigida × P. taeda. Five embryogenic lines were obtained using a modified P. taeda basal medium, and the highest initiation rate was 0.55%, for seeds collected in 2 weeks, between July 3 and 16. Histological observation revealed that zygotic embryos of those seeds were mostly at the proembryonic stage or in transition to precotyledonary stages. For the successful maturation of somatic embryos, abscisic acid and gellan gum were needed in the medium. The results show that, although further tests and development are required, somatic embryogenesis could provide a viable option for propagating P. rigida × P. taeda hybrids.

Construction of Conditional Acid Ceramidase Knockout Mice and in vivo Effects on Oocyte Development and Fertility

The number of resting follicles in the ovary and their successful maturation during development define the fertile female lifespan. Oocytes, enclosed within follicles, are subject to natural selection, and the majority will undergo apoptosis during prenatal life through adulthood. Our previous studies revealed high levels of the lipid hydrolase, acid ceramidase (AC), in human and mouse oocytes, follicular fluid and cumulus cells. In addition, supplementation of in vitro fertilization media with recombinant AC enhanced the survival of oocytes and preimplantation embryos. Herein we constructed and used a conditional knockout mouse model of AC deficiency (cACKO) to further investigate the role of this enzyme in oocyte survival in vivo. Immunohistochemical staining, activity assays, and western blot analysis revealed that AC expression was high in the ovaries of normal mice, particularly in the theca cells. After induction of the AC gene knockout with tamoxifen (TM), AC levels decreased in ovaries, and ceramide was correspondingly elevated. A novel immunostaining method was developed to visualize follicles at various stages, and together with light microscopic examination, the transition of the follicle from the secondary to antral stage was found to be defective in the absence of AC. Western blot analysis showed elevated BAX and PARP expression in TM-treated cACKO mouse ovaries compared to control animals. In parallel, the levels of BCL-2 and anti-Mullerian hormone, a marker of ovarian reserve, were decreased. In addition to the above, there was a significant decrease in fertility observed in the TM-treated cACKO mice. Together, these data suggest that AC plays an important role in the preservation of fertility by maintaining low ceramide levels and preventing apoptosis of theca cells, thereby promoting survival of the follicle during the transition from the secondary to antral stage.

Temporal expression of marker transcripts: Key to successful maturation and development of mammalian oocytes

In vitro embryo production (IVEP) is one of the tool to gain rapid genetic improvement in livestock besides its use in conservation of endangered species. IVEP provides necessary starting material for the basic research in animal development, embryo genomics and stem cells. The success of IVEP depends upon several major factors such as quality of oocyte and sperms, type of culture medium, culture conditions etc. In vitro produced embryos differ substantially from in vivo derived embryos in the mRNA expression patterns of genes important for development. As female germplasm (oocyte) is more valuable when compared to male germplasm (sperm) because of scanty availability and relatively fixed number, it is essential to maintain quality of oocyte in IVEP. Oocyte maturation is an inevitable step towards successful IVEP as the process of maturation is still not clearly revealed, it is essential to identify temporal expression pattern of marker genes involved during maturation process for production of developmentally competent embryos. Mammalian models can provide better insight into the oocyte maturation mechanism. This review focuses on the various marker genes which are essential for successful maturation of mammalian oocyte and are key regulators in deciding the fate of oocytes for production of developmentally competent embryos.

197 INHIBITION OF CYSTEINE PROTEINASES DURING IN VITRO OOCYTE MATURATION IMPROVES DEVELOPMENT OF PORCINE CUMULUS - OOCYTE COMPLEXES

Biochemical differences exist between oocytes that give rise to viable blastocysts and oocytes that give rise to embryos that are developmentally compromised. For example, specific proteolytic enzymes (e.g. cathepsin B) are transcriptionally abundant in in vitro-matured bovine oocytes from prepubertal heifers that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus–oocyte complexes (COC) that represented both poor- and good-quality oocytes. Those reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development. This project sought to determine if similar results would be obtained in a porcine oocyte/embryo culture system. Inclusion of 10 and 20 μM E-64 in maturation medium was performed. Maturation rates of porcine COC in 20 μM E-64 were elevated compared to those incubated in 10 μM E-64 (74% vs 53%; P < 0.05) or without E-64 (55%; P < 0.05: N = 1750 oocytes tested). Successful maturation to metaphase II was based on the presence of a polar body and a uniform cytoplasm 44 h after follicular aspiration. Based on these preliminary results and the earlier bovine work, it was hypothesized that the E-64 was having little influence on normal oocytes, but was promoting maturation of low-quality oocytes, possibly those that were beginning to degenerate. Consequently, 20 μM of E-64 was added to the maturation media of COC segregated based on morphological characteristics of the oocytes. Good COC had a homogeneous cytoplasm and greater than 3 layers of cumulus cells; the COC were considered poor if they displayed a nonhomogeneous cytoplasm and 1 layer or less of cumulus cells, yet were still considered fertilizable. Without E-64, an increase in maturation was measured when good oocytes were compared to poor oocytes (52% vs 29%; P < 0.05: N = 1600). No significant differences in maturation were observed between good oocytes incubated in the presence or absence of E-64. Likewise, no significant differences were observed between poor oocytes incubated in the presence or absence of E-64. The percentage of maturation of good oocytes cultured in E-64 was significantly higher than that of poor oocytes cultured with E-64 (67% vs 43%; P < 0.05). Maturation with the inhibitor did not significantly affect the subsequent cleavage or blastocyst rates of embryos that arose from these oocyte groups after fertilization. These experiments suggest that inhibition of cysteine proteinases significantly promotes oocyte maturation, as was seen in previous bovine work. Our data did not support the hypothesis that cysteine proteinase inhibition was selectively improving maturation of poor oocytes within the pool. It remains possible that increased maturation in good oocytes is a result of cysteine inhibition on juvenile oocytes that morphologically appeared good and the effect was less on already degenerated oocytes that appeared poor. Differences between treatments were determined by ANOVA with post-test by Tukey's multiple comparison test.

Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity

In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.

Involvement of oxidative stress and autointoxication in leaf senescence of Amomum villosum

To clarify whether oxidative stress and autointoxication were involved in leaf senescence of Amomum villosum, the successive leaf maturation and senescence were characterized first, and then the effects of potential autointoxicants on attached mature leaf were determined in this study. Maximum photosynthetic rates (P max) decreased greatly in senescent leaves, which were associated with the decreases in the contents of soluble proteins and chlorophyll (Chl), chlorophyll a/b and stomatal conductance. Maximum efficiency of photosystem (PS) II photochemistry (F v/F m), photochemical quenching coefficient and quantum yield of PS II (Φ PSII) decreased much less in senescent leaves than P max, leading to high PS II excitation pressures. The activities of peroxidase, ascorbate peroxidase and superoxide dismutase increased in senescent leaves, while catalase activity and non-photochemical quenching coefficient decreased. The high PS II excitation pressures in senescent leaves were not effectively dealt with, leading to oxidative stress, as indicated by high malondialdehyde content. Aqueous extracts of leaves and roots of A. villosum and topsoils under this plant inhibited P max more greatly than F v/F m and Φ PSII, leading to high PS II excitation pressures in mature leaves of A. villosum grown in nutrient solution containing the aqueous extracts, and therefore to oxidative stress. Our results indicate that oxidative stress is involved in leaf senescence in A. villosum and autointoxication exacerbates leaf senescence by increasing oxidative stress.

Radical reversal of vasoactive intestinal peptide (VIP) receptors during early lymphopoiesis

Successful thymocyte maturation is essential for normal, peripheral T cell function. Vasoactive intestinal peptide (VIP) is a neuropeptide which is highly expressed in the thymus that has been shown to modulate thymocyte development. VIP predominantly binds two G protein coupled receptors, termed vasoactive intestinal peptide receptor 1 (VPAC1) and VPAC2, but their expression profiles in CD4−/CD8− (double negative, DN) thymocyte subsets, termed DN1–4, have yet to be identified. We hypothesized that a high VPAC1:VPAC2 ratio in the earliest thymocyte progenitors (ETP cells) would be reversed during early lymphopoiesis as observed in activated, peripheral Th2 cells, as the thymus is rich in Th2 cytokines. In support of this hypothesis, high VPAC1 mRNA levels decreased 1000-fold, accompanied with a simultaneous increase in VPAC2 mRNA expression during early thymocyte progenitor (ETP/DN1)→DN3 differentiation. Moreover, arrested DN3 cells derived from an Ikaros null mouse (JE-131 cells) failed to completely reverse the VIP receptor ratio compared to wild type DN3 thymocytes. Surprisingly, VPAC2−/− mice did not show significant changes in relative thymocyte subset numbers. These data support the notion that both VPAC1 and VPAC2 receptors are dynamically regulated by Ikaros, a master transcriptional regulator for thymocyte differentiation, during early thymic development. Moreover, high VPAC1 mRNA is a novel marker for the ETP population making it enticing to speculate that the chemotactic VIP/VPAC1 signaling axis may play a role in thymocyte movement. Also, despite the results that VPAC2 deficiency did not affect thymic subset numbers, future studies are necessary to determine whether downstream T cell phenotypic changes manifest themselves, such as a propensity for a Th1 versus Th2 polarization.

A novel marsupial pri-miRNA transcript has a putative role in gamete maintenance and defines a vertebrate miRNA cluster paralogous to the miR-15a/miR-16-1 cluster

Successful maintenance, survival and maturation of gametes rely on bidirectional communication between the gamete and its supporting cells. Before puberty, factors from the gamete and its supporting cells are necessary for spermatogonial stem cell and primordial follicle oocyte maintenance. Following gametogenesis, gametes rely on factors and nutrients secreted by cells of the reproductive tracts, the epididymis and/or oviduct, to complete maturation. Despite extensive studies on female and male reproduction, many of the molecular mechanisms of germ cell maintenance remain relatively unknown, particularly in marsupial species. We present the first study and characterisation of a novel primary miRNA transcript, pri-miR-16c, in the marsupial, the stripe-faced dunnart. Bioinformatic analysis showed that its predicted processed miRNA - miR-16c - is present in a wide range of vertebrates, but not eutherians. In situ hybridisation revealed dunnart pri-miR-16c expression in day 4 (primordial germ cells) and day 7 (oogonia) pouch young, in primary oocytes and follicle cells of primordial follicles but then only in follicle cells of primary, secondary and antral follicles in adult ovaries. In the adult testis, pri-miR-16c transcripts were present in the cytoplasm of spermatogonial cells. The oviduct and the epididymis both showed expression, but not any other somatic tissues examined or conceptuses during early embryonic development. This pattern of expression suggests that pri-miR-16c function may be associated with gamete maintenance, possibly through mechanisms involving RNA transfer, until the zygote enters the uterus at the pronuclear stage.

Efficiency of the kidney disease outcomes quality initiative guidelines for preemptive vascular access in an academic setting

The National Kidney Foundation-Kidney Diseases Outcomes Quality Initiative (KDOQI) for vascular access guidelines state that patients with late-stage chronic kidney disease (CKD) should undergo native arteriovenous fistula (AVF) creation at least 6 months before anticipated start of hemodialysis (HD) treatments to obviate the need for other vascular access types, such as grafts or central catheters. The objective of this study was to determine the incidence of HD, the functional patency, and associated morbidity of AVFs in patients with late-stage CKD placed according to KDOQI. Consecutive patients with late-stage CKD who underwent AVF creation using KDOQI guidelines for anatomy between January 2003 and December 2007 at two tertiary academic centers were retrospectively evaluated. Baseline demographics, AVF type, and clinical comorbidities were recorded. Patients were stratified into one of four groups (groups A-D) over the follow-up course based on two end points: patency of their AVF and whether or not they began HD. The ideal primary outcome was AVF maturation and use for HD (group A; cumulative functional patency). Other outcomes included AVF patency but no HD (group B), HD with AVF failure (group C), or no HD and AVF abandonment (ie, death, refused hemodialysis, kidney transplant, or fistula failure; group D). Secondary outcomes were time to first cannulation, complications, and secondary interventions. AVFs were created (46% forearm and 54% upper arm) in 150 patients with CKD (85% men, median age 63 years old). At a median follow-up of 10 months, 74 patients (49%) were receiving HD and of these, 48 patients (65%) were using their AVF (group A), whereas 26 patients (35%) were not due to fistula failure (group C). Thirty-four patients (23%) never initiated HD treatments, but had a viable AVF (group B), and 42 patients (28%) never initiated HD and abandoned their AVF (group D). Overall, AVF abandonment was 51%. Mean maturation time of all AVFs successfully cannulated was 285 days (range, 30-1265 days). Complications encountered were maturation failure for cannulation (15%), focal stenosis requiring intervention (13%), inadequate flows on HD (9%), steal syndrome (9%), and thrombosis (8%). Cumulative functional patency for all AVFs was 19% and 27% at 6 and 12 months, respectively, with a mean number of two interventions per AVF (range, 1-10). Mortality during the study was 23%. Despite successful creation and maturation of a preemptive AVF in nearly two-thirds of patients who started HD during the follow-up and given the following observations: the high overall mortality of the population, the morbidity and costs in secondary procedures of AVF creation, and the high incidence of abandonment, it is unclear if this strategy would demonstrate a benefit in a randomized trial when compared to other access strategies.

Independent Prediction Factors for Primary Patency Loss in Arteriovenous Grafts within Six Months

The objective of this study was to explore the association between loss of primary functional patency within 6 months of first use and demographic and clinical characteristics in patients with arteriovenous grafts (AVGs) receiving chronic hemodialysis. The knowledge and management of these characteristics will minimize the proportion of catheterdependent dialysis patients for whom AVGs are the best choice. This was a retrospective study of all chronic hemodialysis patients with AVGs followed by the Southern Alberta Renal Program from January 2005 to June 2008. Demographic and clinical variables and initial intra-access blood flow (IABF) were compared between those with and without loss of primary functional patency. To determine the contribution of independent variables to the dependant variable of loss of primary functional patency, a multivariable analysis using logistic regression was performed. The incidence of primary failure was 30% (107/359). Multivariable analysis found that low initial IABF (<650 mL/ min, odds ratio [OR] 31, P < 0.001), presence of diabetes (OR 3.5, P = 0.001), older age (>65 years OR 3.2, P< 0.001), and presence of peripheral vascular disease (OR 2.5, P< 0.005) were independently associated with loss of primary patency. AVGs are sometimes a better choice for those patients in which the time to and probability of successful fistula maturation may be a concern. Close monitoring of AVGs in patients with the identified risk factors associated with loss of primary patency may improve the life expectancy of the access.

Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation

Many vascular surgeons construct arteriovenous fistulas (AVFs) for hemodialysis access as the primary choice access. A significant number of AVFs fail to mature, however, leading to patient frustration and repeated operations. Metalloproteinase (MMP) activity, particularly MMP-2 and MMP-9, may be important for AVF maturation. We therefore sought to identify whether serum MMP levels could serve as a biomarker for predicting future successful AVF maturation. Blood was collected from patients with chronic renal insufficiency at the time of surgery for long-term hemodialysis access. Serum was separated from whole blood and ultracentrifuged at 1000g for 10 minutes. Serum aliquots were frozen at -80°C until used for analysis. Enzyme-linked immunosorbent assay was used to assay levels of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase type 2 (TIMP-2), and TIMP type 4 (TIMP-4). Clinical end points were used to divide patients into failed and matured AVF groups. Successful maturation was considered in patients who had specific duplex findings or 1 month of successful two-needle cannulation hemodialysis. MMP/TIMP ratios were calculated as an index of the MMP axis activity because MMP activity parallels alterations in TIMP levels. Of 20 enrolled patients, AVF maturation was successful in 13 and failed in 7. Serum levels of MMP-2/TIMP-2 were significantly higher in patients with matured AVFs vs levels in those that failed (P = .003). Similarly, a trend toward increased serum levels of MMP-9/TIMP-4 was found in patients with successful AVF (P = .06). MMP-2 and TIMP-2 levels were different among patients whose AVF matured vs those who did not. Further follow-up studies to determine the predictability of AVF maturation using relative patient serum levels of MMP-2 and TIMP-2 should be performed.

Measuring the Hierarchy of Molecular Events During Clathrin‐Mediated Endocytosis

A well-orchestrated hierarchy of molecular events is required for successful initiation and maturation of clathrin-coated pits (CCPs). Nevertheless, CCPs display a broad range of lifetimes. This dynamic heterogeneity could either reflect differences in the temporal hierarchy of molecular events, or similar CCP maturation processes with variable kinetics. To address this question, we have used multi-channel image acquisition and automated analysis of CCP dynamics in combination with a new method to quantify the time courses of recruitment of endocytic factors to CCPs of different lifetimes. Using this approach we have extracted the kinetics of recruitment and disassembly of fluorescently labeled clathrin and/or AP-2 throughout the entire lifetime of temporally defined CCP cohorts. On the basis of these analyses, we can (i) directly correlate recruitment profiles of these two proteins; (ii) define five distinct CCP maturation phases, i.e. initiation, growth, maturation, separation and departure; (iii) distinguish events with absolute versus fractional timing and (iv) provide information on the spatial distribution of fluorophores during CCP maturation. Emerging from these analyses is a more clearly defined role for AP-2 in determining the temporal hierarchy for clathrin recruitment and CCP maturation. This method provides a new means to identify other such hierarchies during CCP maturation.

Use of Underground Brackish Water for Reproduction and Larviculture of Rainbow Trout, Oncorhynchus mykiss

Broodfish were reared for 6 months in underground brackish water (11 ppt), and the quality of eggs and semen was evaluated in comparison to broodfish held in fresh water. Fry produced by the broodfish in brackish water (S) and freshwater (F) were then reared in different salinities (1, 4, 7, and 10 ppt). Broodfish successfully matured and spawned in underground brackish water, and gonad quality was better than of the broodfish in fresh water (p < 0.01). Growth indices of fry produced by both groups decreased with increasing salinity (p < 0.01). Survival was not affected by the salinities tested. Underground brackish water can be used as a source to induce successful sexual maturation and to produce high-quality gonads.

Too short to spawn? Implications of small body size and swimming distance on successful migration and maturation of the European eel Anguilla anguilla

Individual net fat reserves after migration and reproductive investments were calculated for migrating female silver eels Anguilla anguilla (n = 387) collected in the outlet region of the Baltic Sea during the autumn run. It is estimated that 20·4% of the A. anguilla had completely exhausted all initial fat reserves and that 45·0% of A. anguilla were within 90% of complete energy depletion after migration and reproduction. This study concludes that a combination of body size and distance (6900 km) to the spawning area in the Sargasso Sea explains the results. An increase in the costs of migration due to heavy infection with Anguillicoloides crassus was also evaluated in an additional scenario with results showing that 26·4% of the A. anguilla had completely depleted all fat reserves. It is hypothesized that a large proportion of female silver A. anguilla from the Baltic Sea catchment area will have inadequate or suboptimal reserves for successful migration and reproduction.

Toll-Like Receptor 4 Activation in Cancer Progression and Therapy

Cancer immunotherapy has been the focus of intense research since the late 19th century when Coley observed that bacterial components can contribute to cancer regression by eliciting an antitumor immune response. Successful activation and maturation of tumor-specific immune cells is now known to be mediated by bacterial endotoxin, which activates Toll-like receptor 4 (TLR4). TLR4 is expressed on a variety of immune as well as tumor cells, but its activation can have opposing effects. While TLR4 activation can promote antitumor immunity, it can also result in increased tumor growth and immunosuppression. Nevertheless, TLR4 engagement by endotoxin as well as by endogenous ligands represents notable contribution to the outcome of different cancer treatments, such as radiation or chemotherapy. Further research of the role and mechanisms of TLR4 activation in cancer may provide novel antitumor vaccine adjuvants as well as TLR4 inhibitors that could prevent inflammation-induced carcinogenesis.

Differentiation and function of human monocyte-derived dendritic cells under the influence of leflunomide

Leflunomide is an immunosuppressive drug effective in experimental models of transplantation and autoimmune diseases and in the treatment of active rheumatoid arthritis (RA). Having in mind that it has been shown that some other immunosuppressive drugs (glucocorticoids, mycophenolate mofetil, sirolimus etc.) impair dendritic cell (DC) phenotype and function, we investigated the effect of A77 1726, an active metabolite of leflunomide, on the differentiation and function of human monocyte-derived dendritic cells (MDDC) in vitro. Immature MDDC were generated by cultivating monocytes in medium supplemented with GM-CSF and IL-4. To induce maturation, immature MDDC were cultured for 2 additional days with LPS. A77 1726 (100 ?M) was added at the beginning of cultivation. Flow cytometric analysis showed that MDDC differentiated in the presence of A77 1726 exhibited an altered phenotype, with a down-regulated surface expression of CD80, CD86, CD54 and CD40 molecules. Furthermore, the continuous presence of A77 1726 during differentiation and maturation prevented successful maturation, judging by the decreased expression of maturation marker CD83, costimulatory and adhesive molecules on A77 1726-treated mature MDDC. In addition, A77 1726-pretreated MDDC exhibited a poor stimulatory capacity of the allogeneic T cells and a low production of IL-10 and IL-18. These data suggest that leflunomide impairs the differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

Management Of The Immature Autogenous Arteriovenous Fistula

A high-quality autogenous arteriovenous fistula provides the optimal access for hemodialysis. Following initial surgical construction of a fistula, the maturation process is driven by hemodynamic, cellular, and humoral factors that must result in increased blood flow, vessel dilation, and thickening of the vessel wall before the fistula can be successfully used for dialysis needle access. Different demands are placed on each fistula depending on the individual patient's hemodialysis requirements, which must be clearly understood to properly assess and treat the immature fistula. When spontaneous maturation fails to achieve a functional fistula, additional surgical or minimally invasive interventional procedures may be necessary to enhance the maturation process. Various techniques have been reported to achieve successful fistula maturation. The purpose of this article is to review the concepts of fistula maturation and the interventions that may be performed in cases where there is failure to mature spontaneously.

Activation of Phosphatidylinositol 3-Kinase/Akt and Impairment of Nuclear Factor-κB: Molecular Mechanisms Behind the Arrested Maturation/Activation State of Leishmania infantum-Infected Dendritic Cells

Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.

Microanatomía de los testisacos y espermatogénesis de Helobdella michaelseni (Hirudinea, Glossiphoniidae)

A detailed preliminary histological analysis of Helobdella michaelseni Blanchard 1900 testisacs is described. The male reproductive system is composed of seven pairs of spherical testisacs found intersegmentally from somites XIII/XIV to somites XIX/XX. Spermiducts reach the back of somites XVII/ XVIII or XIX/XX. Spindle form atria. Male pore at somite XII a1/a2. The spermatogenesis occurs in the lu men of fluid-filled coelomic sacs. Each testisacs is clothed by mesotelium. Inside it, the germinal cells are connected to the cytophore and develop functional units called polyplasts. The spermatozoo are released into testisacs after the cytophore reabsorption. Five stages of spermatogenesis are described taking into account the successive maturation stages of germinal cells and the changes in the cytophore. Key words: microanatomy, testisacs, Helobdella michaelseni, spermatogenesis.

Artificial maturation, fertilization, and early development of the American eel (Anguilla rostrata)

Spawning for the American eel ( Anguilla rostrata (Le Sueur, 1817)) takes place in secretive locations within the Sargasso Sea, which has thus far prevented investigations of gametogenesis and early development in this ecologically and commercially important species. Attempts to induce maturation and reproduction in this species have been few and have produced limited results, with a single report of the production of gastrula-stage embryos. Here we report the successful maturation of female American eels. Maturation occurred within 13 weeks and ovulation was induced with a single injection of 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Following in vitro fertilization, embryogenesis through hatching was observed and larvae were maintained for up to 6 days. We show that a crucial factor for successful fertilization is the stage of the oocyte at the time of induced ovulation. Oocytes that had not reached the migratory nucleus stage, or had passed this stage, were not successfully fertilized. These findings demonstrate that American eel can reproduce in the laboratory and previously untestable hypotheses pertaining to the developmental biology of this elusive species can now be explored.