ABSTRACT
Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.
Subject(s)
RNA Polymerase II , Semen , Male , Humans , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Semen/metabolism , Meiosis/genetics , Spermatogenesis/genetics , Gene ExpressionABSTRACT
Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to mature spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double strand break formation by SPO11, and disruption of SPO11 expression in germ cells lacking NELF.
ABSTRACT
The central dilemma in treating patients with refractory or relapsed classical Hodgkin lymphoma (RRHL) is the developed resistance to chemotherapy. In recent years, significant advances have been made with the introduction of targeted immunotherapy such as brentuximab vedotin (BV) and nivolumab (NV). As monotherapy, BV and NV have demonstrated high response rates but with an opportunity for disease progression. In other studies, BV or NV is given in combination with chemotherapy as a bridge to hematopoietic stem cell transplantation for curative therapy. This review will investigate the effect of BV and NV as single agents, in combination with each other, or given concurrently with chemotherapy on the response and survival rate of patients with RRHL.
ABSTRACT
BACKGROUND: Development of pretransplantation islet culture strategies that preserve or enhance ß-cell viability would eliminate the requirement for the large numbers of islets needed to restore insulin independence in type 1 diabetes patients. We investigated whether glial cell line-derived neurotrophic factor (GDNF) could improve human islet survival and posttransplantation function in diabetic mice. METHODS: Human islets were cultured in medium supplemented with or without GDNF (100 ng/mL) and in vitro islet survival and function assessed by analyzing ß-cell apoptosis and glucose stimulated insulin release. In vivo effects of GDNF were assessed in streptozotocin-induced diabetic nude mice transplanted under the kidney capsule with 2000 islet equivalents of human islets precultured in medium supplemented with or without GDNF. RESULTS: In vitro, human islets cultured for 2 to 10 days in medium supplemented with GDNF showed lower ß-cell death, increased Akt phosphorylation, and higher glucose-induced insulin secretion than islets cultured in vehicle. Human islets precultured in medium supplemented with GDNF restored more diabetic mice to normoglycemia and for a longer period after transplantation than islets cultured in vehicle. CONCLUSIONS: Our study shows that GDNF has beneficial effects on human islet survival and could be used to improve islet posttransplantation survival.
Subject(s)
Diabetes Mellitus, Experimental/surgery , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Graft Survival/drug effects , Insulin-Secreting Cells/drug effects , Islets of Langerhans Transplantation/methods , Islets of Langerhans/drug effects , Animals , Apoptosis/drug effects , Cells, Cultured , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/metabolism , Disease Models, Animal , Glucose/pharmacology , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Mice , Mice, Nude , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Streptozocin/adverse effects , Transplantation, HeterologousABSTRACT
The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-beta) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and beta-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP(+)) compared with those cultured with MPP(+) alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-beta signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Catecholamines/metabolism , Cell Differentiation/physiology , Enteric Nervous System/cytology , Neurons/cytology , Nitrergic Neurons/cytology , Smad1 Protein/metabolism , 1-Methyl-4-phenylpyridinium/pharmacology , Active Transport, Cell Nucleus/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Cells, Cultured , Gene Expression/drug effects , Gene Expression/genetics , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Mice , Neurofilament Proteins/genetics , Neurons/drug effects , Neurons/metabolism , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Nitrergic Neurons/metabolism , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type I/metabolism , Phosphorylation/drug effects , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Smad1 Protein/genetics , Tubulin/genetics , Tubulin/metabolism , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Ubiquitin Thiolesterase/metabolismABSTRACT
BACKGROUND & AIMS: The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) promoter element. METHODS: Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees C in the presence of glial cell line-derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS(-/-) mice. RESULTS: Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, tau, synaptic marker synaptophysin, characteristic receptors of enteric neurons, Ret, and 5-hydroxytryptamine-receptor subtypes at 33 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees C. Glial cell line-derived neurotrophic factor resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS(-/-) mice colon improved colonic motility. CONCLUSIONS: We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung's disease.
