Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro.

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

Cryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT) mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopreserved oocyte cytoplasm (SCNT-CROC) was inferior to those using fresh cytoplasm (SCNT-FOC). Using RNA-sequencing analysis, we found upregulation of eight pro-apoptotic-related genes (Cyct, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, and Pmaip) in the SCNT-CROC group. Furthermore, the addition of melatonin, an agent that reduces apoptosis and ROS production, enhanced blastocyst formation rates in the SCNT-CROP group when compared with the melatonin-untreated group. Additionally, melatonin treatment increased the derivation efficiency of pluripotent stem cells from cloned embryos using cryopreserved oocyte.

Hypergravity hinders axonal development of motor neurons in Caenorhabditis elegans.

As space flight becomes more accessible in the future, humans will be exposed to gravity conditions other than our 1G environment on Earth. Our bodies and physiology, however, are adapted for life at 1G gravity. Altering gravity can have profound effects on the body, particularly the development of muscles, but the reasons and biology behind gravity's effect are not fully known. We asked whether increasing gravity had effects on the development of motor neurons that innervate and control muscle, a relatively unexplored area of gravity biology. Using the nematode model organism Caenorhabditis elegans, we examined changes in response to hypergravity in the development of the 19 GABAergic DD/VD motor neurons that innervate body muscle. We found that a high gravity force above 10G significantly increases the number of animals with defects in the development of axonal projections from the DD/VD neurons. We showed that a critical period of hypergravity exposure during the embryonic/early larval stage was sufficient to induce defects. While characterizing the nature of the axonal defects, we found that in normal 1G gravity conditions, DD/VD axonal defects occasionally occurred, with the majority of defects occurring on the dorsal side of the animal and in the mid-body region, and a significantly higher rate of error in the 13 VD axons than the 6 DD axons. Hypergravity exposure increased the rate of DD/VD axonal defects, but did not change the distribution or the characteristics of the defects. Our study demonstrates that altering gravity can impact motor neuron development.

NGT-3D: a simple nematode cultivation system to study Caenorhabditis elegans biology in 3D.

The nematodeCaenorhabditiselegansis one of the premier experimental model organisms today. In the laboratory, they display characteristic development, fertility, and behaviors in a two dimensional habitat. In nature, however,C. elegansis found in three dimensional environments such as rotting fruit. To investigate the biology ofC. elegansin a 3D controlled environment we designed a nematode cultivation habitat which we term the nematode growth tube or NGT-3D. NGT-3D allows for the growth of both nematodes and the bacteria they consume. Worms show comparable rates of growth, reproduction and lifespan when bacterial colonies in the 3D matrix are abundant. However, when bacteria are sparse, growth and brood size fail to reach levels observed in standard 2D plates. Using NGT-3D we observe drastic deficits in fertility in a sensory mutant in 3D compared to 2D, and this defect was likely due to an inability to locate bacteria. Overall, NGT-3D will sharpen our understanding of nematode biology and allow scientists to investigate questions of nematode ecology and evolutionary fitness in the laboratory.

An integrated systems biology approach identifies positive cofactor 4 as a factor that increases reprogramming efficiency.

Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes.

Violacein: Properties and Production of a Versatile Bacterial Pigment.

Violacein-producing bacteria, with their striking purple hues, have undoubtedly piqued the curiosity of scientists since their first discovery. The bisindole violacein is formed by the condensation of two tryptophan molecules through the action of five proteins. The genes required for its production, vioABCDE, and the regulatory mechanisms employed have been studied within a small number of violacein-producing strains. As a compound, violacein is known to have diverse biological activities, including being an anticancer agent and being an antibiotic against Staphylococcus aureus and other Gram-positive pathogens. Identifying the biological roles of this pigmented molecule is of particular interest, and understanding violacein's function and mechanism of action has relevance to those unmasking any of its commercial or therapeutic benefits. Unfortunately, the production of violacein and its related derivatives is not easy and so various groups are also seeking to improve the fermentative yields of violacein through genetic engineering and synthetic biology. This review discusses the recent trends in the research and production of violacein by both natural and genetically modified bacterial strains.

A role for peroxidasin PXN-1 in aspects of C. elegans development.

The Caenorhabditis elegans peroxidasins, PXN-1 and PXN-2, are extracellular peroxidases; pxn-2 is involved in muscle-epidermal attachment during embryonic morphogenesis and in specific axon guidance. Here we investigate potential roles of the other homologue of peroxidasin, pxn-1, in C. elegans. A pxn-1 deletion mutant showed high lethality under heat-stress conditions. Using a transcriptional GFP reporter, pxn-1 expression was observed in various tissues including neurons, muscles, and hypodermis. A translational fusion showed that PXN-1::GFP was secreted and localized in extracellular matrix, particularly along body wall muscles and pharyngeal muscles. Various neuronal developmental defects were observed in pxn-1 mutants and in pxn-1 over-expressing animals, including handedness, branching, breakage, tangling, and defasciculation. These results suggest that pxn-1, like other peroxidasins, plays an important role throughout development.

DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction.

OBJECTIVE: To determine whether ligation-mediated real-time polymerase chain reaction (LM-RT-PCR), which combines LM-PCR, and RT-PCR, can detect sperm DNA fragmentation (DF) in human semen samples. DESIGN: Three-way comparison of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD), and LM-RT-PCR for detecting sperm DNA fragmentation. SETTING: University hospital-based research laboratory. PATIENT(S): Twenty-five men presenting at an infertility clinic. INTERVENTION(S): Basic analysis of sperm concentration, motility, vitality, and morphology, with each semen sample equally divided into three aliquots that were evaluated for fragmentation using TUNEL, SCD, and LM-RT-PCR assays. MAIN OUTCOME MEASURE(S): In TUNEL and SCD assays, counts of the number of sperm with tetramethylrhodamine (TMR) red signals or no halo; in LM-RT-PCR results, evaluation of the threshold cycles (Ct) and relative fluorescence unit (RFU) values. RESULT(S): The median percentage of sperm with positive results for fragmentation in the TUNEL and SCD assays were 20.5% and 20.7%, respectively. To compare the accuracy of the TUNEL, SCD, and LM-RT-PCR assays, we divided the semen samples into two groups according to the TUNEL results: low and high percentage of sperm fragmentation. In the LM-RT-PCR results, the values of the cycles of threshold (Ct) and relative fluorescence unit (RFU) statistically significantly differed between the low and high percentage of sperm fragmentation groups. Comparisons among the TUNEL, SCD, and LM-RT-PCR assays revealed that the correlation patterns according to DNA fragmentation were similar in both the groups with high and low percentage of DNA fragmentation. Our morphologic analysis indicated that the fragmentation of sperm DNA did not appear to influence sperm morphology. CONCLUSION(S): These results indicate that the LM-RT-PCR technique is another useful tool for detecting DNA fragmentation, a parameter of sperm quality in human semen alone or combined with TUNEL or SCD assays.

The multiple faces of calcineurin signaling in Caenorhabditis elegans: development, behaviour and aging.

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca2+-calmodulin-dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.

Two thioredoxin reductases, trxr-1 and trxr-2, have differential physiological roles in Caenorhabditis elegans.

Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide reductase family, which mainly functions in the thioredoxin system. TrxR is found in all living organisms and exists in two major ubiquitous isoenzymes in higher eukaryotic cells; One is cytosolic and the other mitochondrial. Mitochondrial TrxR functions to protect mitochondria from oxidative stress, where reactive oxidative species are mainly generated, while cytosolic TrxR plays a role to maintain optimal oxido-reductive status in cytosol. In this study, we report differential physiological functions of these two TrxRs in C. elegans. trxr-1, the cytosolic TrxR, is highly expressed in pharynx, vulva and intestine, whereas trxr-2, the mitochondrial TrxR, is mainly expressed in pharyngeal and body wall muscles. Deficiency of the non-selenoprotein trxr-2 caused defects in longevity and delayed development under stress conditions, while deletion mutation of the selenoprotein trxr-1 resulted in interference in acidification of lysosomal compartment in intestine. Interestingly, the acidification defect of trxr-1(jh143) deletion mutant was rescued, not only by selenocystein-containing wild type TRXR-1, but also cysteine-substituted mutant TRXR-1. Both trxr-1 and trxr-2 were up-regulated when worms were challenged by environmental stress such as heat shock. These results suggest that trxr-1 and trxr-2 function differently at organismal level presumably by their differential sub-cellular localization in C. elegans.

CNP-1 (ARRD-17), a novel substrate of calcineurin, is critical for modulation of egg-laying and locomotion in response to food and lysine sensation in Caenorhabditis elegans.

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans.

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.

Calcineurin in animal behavior.

The conserved Ca2+/calmodulin-dependent phosphatase calcineurin has been shown to be involved in numerous and diverse functions both at the cellular and organism level. Recent genetic and pharmacological studies in animals including C. elegans, Drosophila, Aplysia, rat and mice have also implicated calcineurin in behavior, particularly in the regulation of plasticity and modulation of behaviors. These studies have not only brought a clearer understanding of the molecular contributions to behavior, but should also give insight into roles that calcineurin may be playing in the cognitive and behavioral defects observed in some diseases.

SHN-1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol-1,4,5-trisphosphate receptor.

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.

The Caenorhabditis elegans homologue of Down syndrome critical region 1, RCN-1, inhibits multiple functions of the phosphatase calcineurin.

A conserved family of calcineurin-regulating proteins whose members have been implicated in several disease models such as Down syndrome, Alzheimer's disease, and cardiac hypertrophy has been identified in several organisms including yeast, mice, and humans. We have characterized Caenorhabditis elegans rcn-1, which belongs to this family of calcineurin regulators, and shows approximately 40% identity with the human homologue DSCR-1. rcn-1 is expressed in hypodermal cells, nerve cords and various neurons, vulva epithelial and muscle cells, marginal cells of the pharynx, and structures of the male tail. rcn-1 expression is upregulated by calcineurin activity. RCN-1 binds to calcineurin A from C.elegans lysate in a calcium-dependent manner, and inhibits bovine calcineurin phosphatase activity dose-dependently. In addition, overexpression of RCN-1 results in calcineurin-deficient phenotypes such as small body size, cuticle defects, fertility defects, slow growth, and serotonin-resistant egg-laying defects. Moreover, phenotypes observed in gain-of-function calcineurin mutant animals were restored to normal by RCN-1 overexpression. These results demonstrate an effective and specific inhibition of calcineurin in vitro as well as in vivo by RCN-1.

Calcineurin, a calcium/calmodulin-dependent protein phosphatase, is involved in movement, fertility, egg laying, and growth in Caenorhabditis elegans.

Calcineurin is a Ca(2+)-calmodulin-dependent serine/threonine protein phosphatase that has been implicated in various signaling pathways. Here we report the identification and characterization of calcineurin genes in Caenorhabditis elegans (cna-1 and cnb-1), which share high homology with Drosophila and mammalian calcineurin genes. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation in the nematode. Calcineurin is expressed in hypodermal seam cells, body-wall muscle, vulva muscle, neuronal cells, and in sperm and the spermatheca. cnb-1 mutants showed pleiotropic defects including lethargic movement and delayed egg-laying. Interestingly, these characteristic defects resembled phenotypes observed in gain-of-function mutants of unc-43/Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and goa-1/G(o)-protein alpha-subunit. Double mutants of cnb-1 and unc-43(gf) displayed an apparent synergistic severity of movement and egg-laying defects, suggesting that calcineurin may have an antagonistic role in CaMKII-regulated phosphorylation signaling pathways in C. elegans.