Miracle-dz/UniFAR-FTUs huggingface dataset:
Atrazine Inhibits Pulsatile Gonadotropin-Releasing Hormone (GnRH) Release Without Altering GnRH Messenger RNA or Protein Levels in the Female Rat1

ABSTRACT Atrazine (ATR) is a commonly used pre-emergence/early postemergence herbicide. Previous work has shown that exposure to high doses of ATR in rats results in blunting of the hormone-induced luteinizing hormone (LH) surge and inhibition of pulsatile LH release without significantly reducing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) agonist. Accompanying the reduction in the LH surge was an attenuation of GnRH neuronal activation. These findings suggest that ATR exposure may be acting to inhibit GnRH release. In this study, we examined GnRH directly to determine the effect of high doses of ATR on GnRH pulsatile release, gene expression, and peptide levels in the female rat. Ovariectomized adult female Wistar rats were treated with ATR (200 mg/kg) or vehicle for 4 days via gavage. Following the final treatment, GnRH release was measured from ex vivo hypothalamic explants for 3 h. In another experiment, animals were administered either vehicle or ATR (50, 100, or 200 mg/kg) daily for 4 days. Following treatment, in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript. Finally, GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue of treated animals. ATR treatment resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis.
Locus-Specific DNA Methylation Reprogramming During Early Porcine Embryogenesis1

ABSTRACT During early mammalian embryogenesis, there is a wave of DNA demethylation postfertilization and de novo methylation around implantation. The paternal genome undergoes active DNA demethylation, whereas the maternal genome is passively demethylated after fertilization in most mammals except for sheep and rabbits. However, the emerging genome-wide DNA methylation landscape has revealed a regulatory and locus-specific DNA methylation reprogramming pattern in mammalian preimplantation embryos. Here we optimized a bisulfite sequencing protocol to draw base-resolution DNA methylation profiles of several selected genes in gametes, early embryos, and somatic tissue. We observed locus-specific DNA methylation reprogramming in early porcine embryos. First, some pluripotency genes (POU5F1 and NANOG) followed a typical wave of DNA demethylation and remethylation, whereas CpG-rich regions of SOX2 and CDX2 loci were hypomethylated throughout development. Second, a differentially methylated region of an imprint control region in the IGF2/H19 locus exhibited differential DNA methylation which was maintained in porcine early embryos. Third, a centromeric repeat element retained a moderate DNA methylation level in gametes, early embryos, and somatic tissue. The diverse DNA methylation reprogramming during early embryogenesis is thought to be possibly associated with the multiple functions of DNA methylation in transcriptional regulation, genome stability and genomic imprinting. The latest technology such as oxidative bisulfite sequencing to identify 5-hydroxymethylcytosine will further clarify the DNA methylation reprogramming during porcine embryonic development.
Semen Parameters Can Be Predicted from Environmental Factors and Lifestyle Using Artificial Intelligence Methods1

ABSTRACT Fertility rates have dramatically decreased in the last two decades, especially in men. It has been described that environmental factors as well as life habits may affect semen quality. In this paper we use artificial intelligence techniques in order to predict semen characteristics resulting from environmental factors, life habits, and health status, with these techniques constituting a possible decision support system that can help in the study of male fertility potential. A total of 123 young, healthy volunteers provided a semen sample that was analyzed according to the World Health Organization 2010 criteria. They also were asked to complete a validated questionnaire about life habits and health status. Sperm concentration and percentage of motile sperm were related to sociodemographic data, environmental factors, health status, and life habits in order to determine the predictive accuracy of a multilayer perceptron network, a type of artificial neural network. In conclusion, we have developed an artificial neural network that can predict the results of the semen analysis based on the data collected by the questionnaire. The semen parameter that is best predicted using this methodology is the sperm concentration. Although the accuracy for motility is slightly lower than that for concentration, it is possible to predict it with a significant degree of accuracy. This methodology can be a useful tool in early diagnosis of patients with seminal disorders or in the selection of candidates to become semen donors.
Xenogeneic Transfer of Adult Quail (Coturnix coturnix) Spermatogonial Stem Cells to Embryonic Chicken (Gallus gallus) Hosts: A Model for Avian Conservation1

ABSTRACT As advanced reproductive technologies have become routine for domesticated species, they have begun to be applied in the field of endangered species conservation. For avian conservation, the most promising technology is the transfer of germ stem cells of exotic species to domestic hosts for the production of gametes. In this study, adult quail (model for exotic species) spermatogonial stem cells were xenogeneically transferred to stages 14–17 chicken host embryos. Fluorescent cellular dyes, quail-specific antibodies, and quail-specific quantitative PCR confirmed donor cell migration to and colonization of the host gonadal ridge. Donor-derived cells were observed by fluorescent microscopy in the caudal area as early as 2 h after injection, in the gonadal ridge at 4 h after injection, as well as in the gonads of stages 35–38 host embryos. Four of eight donor-derived cell flow cytometry-positive host gonads were confirmed by quantitative PCR using quail-specific primers. There was no statistically significant effect of host stage of injection, host gonad isolation stage, or host sex on the number of hosts positive for donor cells or the percent of donor-derived cells per positive gonad. Donor-derived cells isolated from stages 35–38 host gonads costained with the germ stem cell marker SSEA-1, indicating that the donor-derived cells have maintained stem cell-ness. This is the first study to suggest that it is feasible to rescue adult germ stem cells of deceased birds to prolong the reproductive lifespan of critically endangered species or genetically valuable individuals by transferring them to an embryonic chicken host.
SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr)*♦

Evolutionarily conserved structure-selective endonuclease MUS81 forms a complex with EME1 and further associates with another endonuclease SLX4-SLX1 to form a four-subunit complex of MUS81-EME1-SLX4-SLX1, coordinating distinctive biochemical activities of both endonucleases in DNA repair. Viral protein R (Vpr), a highly conserved accessory protein in primate lentiviruses, was previously reported to bind SLX4 to mediate down-regulation of MUS81. However, the detailed mechanism underlying MUS81 down-regulation is unclear. Here, we report that HIV-1 Vpr down-regulates both MUS81 and its cofactor EME1 by hijacking the host CRL4-DCAF1 E3 ubiquitin ligase. Multiple Vpr variants, from HIV-1 and SIV, down-regulate both MUS81 and EME1. Furthermore, a C-terminally truncated Vpr mutant and point mutants R80A and Q65R, all of which lack G2 arrest activity, are able to down-regulate MUS81-EME1, suggesting that Vpr-induced G2 arrest is not correlated with MUS81-EME1 down-regulation. We also show that neither the interaction of MUS81-EME1 with Vpr nor their down-regulation is dependent on SLX4-SLX1. Together, these data provide new insight on a conserved function of Vpr in a host endonuclease down-regulation.
Development of a O-Conotoxin Analogue with Improved Lipid Membrane Interactions and Potency for the Analgesic Sodium Channel NaV1.8

Jennifer R. Deuis, Zoltan Dekan, Marco C. Inserra, Tzong-Hsien Lee, Marie-Isabel Aguilar, David J. Craik, Richard J. Lewis, Paul F. Alewood, Mehdi Mobli , Christina I. Schroeder, Sónia Troeira Henriques, and Irina Vetter From the Institute for Molecular Bioscience and Centre for Advanced Imaging, The University of Queensland, St. Lucia, Queensland 4072, Australia, School of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4102, Australia, and Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia
Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast*

Eukaryotic lipid synthesis is oxygen-dependent with cholesterol synthesis requiring 11 oxygen molecules and fatty acid desaturation requiring 1 oxygen molecule per double bond. Accordingly, organisms evaluate oxygen availability to control lipid homeostasis. The sterol regulatory element-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis in response to changing sterol and oxygen levels. However, notably missing is an SREBP-1 analog that regulates triacylglycerol and glycerophospholipid homeostasis in response to low oxygen. Consistent with this, studies have shown that the Sre1 transcription factor regulates only a fraction of all genes up-regulated under low oxygen. To identify new regulators of low oxygen adaptation, we screened the S. pombe nonessential haploid deletion collection and identified 27 gene deletions sensitive to both low oxygen and cobalt chloride, a hypoxia mimetic. One of these genes, mga2, is a putative transcriptional activator. In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid saturation. Indeed, addition of exogenous oleic acid to mga2Δ cells rescued the observed growth defects. Together, these results establish Mga2 as a transcriptional regulator of triacylglycerol and glycerophospholipid homeostasis in S. pombe, analogous to mammalian SREBP-1.
Mitochondrial Hydrogen Peroxide and Defective Cholesterol Efflux Prevent In Vitro Fertilization by Cryopreserved Inbred Mouse Sperm1

ABSTRACT Recent advances in the cryopreservation of mouse sperm have resulted in dramatically improved in vitro fertilization (IVF) rates, but the biological mechanisms underlying the techniques remain unclear. Two different classes of compounds have been widely utilized to improve the IVF rates of cryopreserved mouse sperm: antioxidants and cyclodextrins. To determine how cryopreservation reduces mouse sperm IVF and how antioxidants and cyclodextrins mitigate this effect, we examined sperm function and oxidative damage after cryopreservation, with and without treatments, in mouse strains important for biomedical research. Our investigation revealed mouse strain-specific effects on IVF by modulation of oxidative stress and cholesterol efflux of cryopreserved sperm. Antioxidants improved IVF rates of C57Bl6/J cryopreserved mouse sperm by reducing hydrogen peroxide produced by sperm mitochondria and ameliorating peroxidative damage to the sperm acrosome. Enhancing cholesterol efflux with cyclodextrin restored capacitation-dependent sperm function and IVF after cryopreservation of C57Bl/6J, C57Bl/6N, and 129X1 mouse sperm. Our results highlight two accessible pathways for continued development of IVF techniques for mouse sperm and provide novel endpoints prognostic of IVF success. These insights may improve sperm cryopreservation methods of other mouse strains and species.
Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics.
Mechanistic Target of Rapamycin (mTOR) Inhibition Synergizes with Reduced Internal Ribosome Entry Site (IRES)-mediated Translation of Cyclin D1 and c-MYC mRNAs to Treat Glioblastoma*

Our previous work has demonstrated an intrinsic mRNA-specific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM.
Administration of Follicle-Stimulating Hormone In Vivo Triggers Testicular Recrudescence of Juvenile European Sea Bass (Dicentrarchus labrax)1

ABSTRACT Follicle-stimulating hormone (Fsh) is thought to act early in the process of spermatogenesis; however, its action in fish has not yet been clearly established. In the present work, we analyzed the effects of recombinant Fsh in sea bass (Dicentrarchus labrax) spermatogenesis according to two different approaches: direct injection of recombinant single-chain Fsh hormone (scFSH) and injection of scFSH coding sequence. Both approaches were efficient in increasing plasma Fsh at 7 and 15 days, respectively, after injection. The Fsh increment caused a significant increase in plasma 11-ketotestosterone levels and induced dramatic changes at the testicular level. Fsh-treated groups showed an increase in germ cell proliferation at Day 7, and cysts of spermatocytes and spermatids were observed at the end of the experiment. After treatment with Fsh, a suppression in amh transcripts and an increase of lhr transcripts were detected at Day 7 and Day 15, respectively, and an increment in fshr expression became evident at Day 23. These results show that Fsh initiates germ cell proliferation, triggering spermatogenesis in sea bass via androgen production and regulation of spermatogenesis-related genes.
Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7*

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.
Modulation of Cell Cycle Progression in the Spermatocyte Cell Line [GC-2spd(ts) Cell-Line] by Cigarette Smoke Condensate (CSC) via Arylhydrocarbon Receptor-Nuclear Factor Erythroid 2-Related Factor 2 (Ahr-Nrf2) Pathway1

ABSTRACT Prior studies in our laboratory have demonstrated that cigarette smoke condensate (CSC) activates arylhydrocarbon receptor (Ahr) leading to upregulation of several antioxidant enzymes in murine spermatocytes. In this study, we show that exposure of the spermatocyte cell line GC-2spd(ts) to CSC induces an increase in Cyp1a1, demonstrating AHR activation, and simultaneous expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), where it is believed to modulate Ahr expression by a feedback mechanism. Pharmacological inhibition by the AHR-antagonist CH223191 and interference by Ahr- and Nrf2-small interfering RNA followed by quantitative real-time PCR implicate the Ahr-Nrf2 pathway in the modulation of DNA damage and growth suppression genes such as Gadd45a and P21 and oxidative stress-related genes Cyp1a1, Nrf2, and Ahrr. Flow cytometry accompanied with cell proliferation assay indicate the CSC induces accumulation of spermatocytes at the S-G2/M phase of the cell cycle. Thus, the data obtained suggest that CSC contains several AHR-agonists that are capable of altering the growth pattern of spermatocytes in vitro through the Ahr-Nrf2 signaling mechanism.
Synergistic Effect of Interferon-Gamma and Tumor Necrosis Factor-Alpha on Coxsackievirus and Adenovirus Receptor Expression: An Explanation of Cell Sloughing During Testicular Inflammation in Mice1

ABSTRACT Coxsackievirus and adenovirus receptor (CAR) is a junction molecule that expresses on Sertoli and germ cells. It mediates Sertoli-germ cell adhesion and facilitates migration of preleptotene/leptotene spermatocytes across the blood–testis barrier, suggesting that CAR-based cell adhesion and migration are crucial for spermatogenesis. Interferon-gamma (IFNG) and tumor necrosis factor alpha (TNF) are two major cytokines that are elevated during testicular inflammation and cause reduced fertility. We investigated the mechanism by which IFNG and TNF exert their disruptive effects on testicular cell adhesion. We have demonstrated that combined treatment with IFNG and TNF (IFNG+TNF) exerts a synergistic effect by downregulating CAR mRNA and protein levels. Immunofluorescence staining revealed that IFNG+TNF treatment effectively removes CAR from the site of cell–cell contact. Using inhibitor and co-immunoprecipitation, we confirmed that IFNG+TNF mediates CAR protein degradation via ubiquitin-proteasome and NFKB pathways. Blockage of ubiquitin-proteasome pathway significantly inhibits CAR degradation, as indicated by the reappearance of CAR at the site of cell–cell contact. Additionally, IFNG+TNF reduces CAR mRNA via transcriptional regulation. Mutational studies have shown that IFNG+TNF-induced CAR repression is achieved by suppression of the basal transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays further confirmed that IFNG+TNF treament not only inhibits binding of the basal transcription factors but also promotes binding of NFKB subunits and Sp1 (negative regulators) to the CAR promoter region. Taken together, IFNG+TNF treatment significantly downregulates CAR expression, which provides an explanation of how cell sloughing in the epithelium mediates, by loss of CAR-based cell adhesion, during testicular inflammation.
CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development *

The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.