Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice

ABSTRACT CUG-BP1/CELF1 is a multifunctional RNA-binding protein involved in the regulation of alternative splicing and translation. To elucidate its role in mammalian development, we produced mice in which the Cugbp1 gene was inactivated by homologous recombination. These Cugbp1 − / − mice were viable, although a significant portion of them did not survive after the first few days of life. They displayed growth retardation, and most Cugbp1 − / − males and females exhibited impaired fertility. Male infertility was more thoroughly investigated. Histological examination of testes from Cugbp1 − / − males showed an arrest of spermatogenesis that occurred at step 7 of spermiogenesis, before spermatid elongation begins, and an increased apoptosis. A quantitative reverse transcriptase PCR analysis showed a decrease of all the germ cell markers tested but not of Sertoli and Leydig markers, suggesting a general decrease in germ cell number. In wild-type testes, CUG-BP1 is expressed in germ cells from spermatogonia to round spermatids and also in Sertoli and Leydig cells. These findings demonstrate that CUG-BP1 is required for completion of spermatogenesis.

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NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

10.1101/2020.09.23.310912 ◽

2020 ◽

Author(s):

Ryuki Shimada ◽

Hiroko Koike ◽

Takamasa Hirano ◽

Yumiko Saga

Keyword(s):

Cell Cycle ◽

Germ Cell ◽

Germ Cells ◽

Rna Binding ◽

Initial Step ◽

Sexually Dimorphic ◽

Wild Type ◽

Male Germ Cells ◽

Cycle Arrest ◽

Male Specific

AbstractDuring murine germ cell development, male germ cells enter the mitotically arrested G0 stage, which is an initial step of sexually dimorphic differentiation. The male specific RNA-binding protein NANOS2 has a key role in suppressing the cell cycle in germ cells. However, the detailed mechanism of how NANOS2 regulates the cell cycle remains unclear. Using single-cell RNA sequencing (scRNA-seq), we extracted the cell cycle state of each germ cell in wild-type and Nanos2-KO testes, and revealed that Nanos2 expression starts in mitotic cells and induces mitotic arrest. We also found that NANOS2 and p38 MAPK work in parallel to regulate the cell cycle, suggesting that several different cascades are involved in the induction of cell cycle arrest. Furthermore, we identified Rheb, a regulator of mTORC1, and Ptma as possible targets of NANOS2. We propose that the repression of the cell cycle is a primary function of NANOS2 and that it is mediated via the suppression of mTORC1 activity by repressing Rheb in a post-transcriptional manner.

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Multi-modal regulation of C. elegans hermaphrodite spermatogenesis by the GLD-1-FOG-2 complex

10.1101/386250 ◽

2018 ◽

Author(s):

Shuang Hu ◽

Lauren E. Ryan ◽

Ebru Kaymak ◽

Lindsay Freeberg ◽

Te-Wen Lo ◽

...

Keyword(s):

Sex Determination ◽

Germ Cell ◽

Germ Cells ◽

Translational Control ◽

Rna Binding ◽

Rna Binding Proteins ◽

Wild Type ◽

C Elegans ◽

Target Mrnas ◽

Mrna Targets

AbstractProper germ cell sex determination in Caenorhabditis nematodes requires a network of RNA-binding proteins (RBPs) and their target mRNAs. In some species, changes in this network enabled limited XX spermatogenesis, and thus self-fertility. In C. elegans, one of these selfing species, the global sex-determining gene tra-2 is regulated in germ cells by a conserved RBP, GLD-1, via the 3’ untranslated region (3’UTR) of its transcript. A C. elegans-specific GLD-1 cofactor, FOG-2, is also required for hermaphrodite sperm fate, but how it modifies GLD-1 function is unknown. Germline feminization in gld-1 and fog-2 null mutants has been interpreted as due to cell-autonomous elevation of TRA-2 translation. Consistent with the proposed role of FOG-2 in translational control, the abundance of nearly all GLD-1 target mRNAs (including tra-2) is unchanged in fog-2 mutants. Epitope tagging reveals abundant TRA-2 expression in somatic tissues, but an undetectably low level in wild-type germ cells. Loss of gld-1 function elevates germline TRA-2 expression to detectable levels, but loss of fog-2 function does not. A simple quantitative model of tra-2 activity constrained by these results can successfully sort genotypes into normal or feminized groups. Surprisingly, fog-2 and gld-1 activity enable the sperm fate even when GLD-1 cannot bind to the tra-2 3’ UTR. This suggests the GLD-1-FOG-2 complex regulates uncharacterized sites within tra-2, or other mRNA targets. Finally, we quantify the RNA-binding capacities of dominant missense alleles of GLD-1 that act genetically as “hyperrepressors” of tra-2 activity. These variants bind RNA more weakly in vitro than does wild-type GLD-1. These results indicate that gld-1 and fog-2 regulate germline sex via multiple interactions, and that our understanding of the control and evolution of germ cell sex determination in the C. elegans hermaphrodite is far from complete.

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Müllerian Inhibiting Substance Is Required for Germ Cell Proliferation during Early Gonadal Differentiation in Medaka (Oryzias latipes)

Endocrinology ◽

10.1210/en.2007-1535 ◽

2007 ◽

Vol 149 (4) ◽

pp. 1813-1819 ◽

Cited By ~ 47

Author(s):

Eri Shiraishi ◽

Norifumi Yoshinaga ◽

Takeshi Miura ◽

Hayato Yokoi ◽

Yuko Wakamatsu ◽

...

Keyword(s):

Cell Proliferation ◽

Germ Cell ◽

Germ Cells ◽

Sex Differentiation ◽

Somatic Cells ◽

Oryzias Latipes ◽

Cell Number ◽

Gonadal Differentiation ◽

Loss Of Function ◽

Germ Cell Proliferation

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-β superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2′-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

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The RNA-binding protein ELAVL1/HuR is essential for mouse spermatogenesis, acting both at meiotic and postmeiotic stages

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0212 ◽

2011 ◽

Vol 22 (16) ◽

pp. 2875-2885 ◽

Cited By ~ 39

Author(s):

Mai Nguyen Chi ◽

Jacques Auriol ◽

Bernard Jégou ◽

Dimitris L. Kontoyiannis ◽

James M.A. Turner ◽

...

Keyword(s):

Germ Cells ◽

Posttranscriptional Regulation ◽

Target Gene ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Female Sterility ◽

Targeted Deletion

Posttranscriptional mechanisms are crucial to regulate spermatogenesis. Accurate protein synthesis during germ cell development relies on RNA binding proteins that control the storage, stability, and translation of mRNAs in a tightly and temporally regulated manner. Here, we focused on the RNA binding protein Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) known to be a key regulator of posttranscriptional regulation in somatic cells but the function of which during gametogenesis has never been investigated. In this study, we have used conditional loss- and gain-of-function approaches to address this issue in mice. We show that targeted deletion of HuR specifically in germ cells leads to male but not female sterility. Mutant males are azoospermic because of the extensive death of spermatocytes at meiotic divisions and failure of spermatid elongation. The latter defect is also observed upon HuR overexpression. To elucidate further the molecular mechanisms underlying spermatogenesis defects in HuR-deleted and -overexpressing testes, we undertook a target gene approach and discovered that heat shock protein (HSP)A2/HSP70-2, a crucial regulator of spermatogenesis, was down-regulated in both situations. HuR specifically binds hspa2 mRNA and controls its expression at the translational level in germ cells. Our study provides the first genetic evidence of HuR involvement during spermatogenesis and reveals Hspa2 as a target for HuR.

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RNA-Binding Protein HuR Regulates Both Mutant and Wild-Type IDH1 in IDH1-Mutated Cancer

Molecular Cancer Research ◽

10.1158/1541-7786.mcr-18-0557 ◽

2018 ◽

Vol 17 (2) ◽

pp. 508-520 ◽

Cited By ~ 4

Author(s):

Mahsa Zarei ◽

Shruti Lal ◽

Ali Vaziri-Gohar ◽

Kevin O'Hayer ◽

Venugopal Gunda ◽

...

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Wild Type

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hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

10.21203/rs.3.rs-1060705/v1 ◽

2021 ◽

Author(s):

Shuiqiao Yuan ◽

Shenglei Feng ◽

Jinmei Li ◽

Hui Wen ◽

Kuan Liu ◽

...

Keyword(s):

Alternative Splicing ◽

Germ Cell ◽

Germ Cells ◽

Rna Binding ◽

Cell Development ◽

Mrna Splicing ◽

Conditional Knockout ◽

Cell Junction ◽

Germ Cell Development ◽

Splicing Regulators

Abstract Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

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Investigating the Roles of RNA Binding Protein Combinations in Neuronal Function and Organismal Behavior

10.25172/jour.4.1.7 ◽

2019 ◽

Vol 4 (Spring 2019) ◽

Author(s):

Alexa Vandenburg

Keyword(s):

Binding Sites ◽

Neuronal Development ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Wild Type ◽

C Elegans ◽

Neuronal Gene ◽

Touch Response

The Norris lab recently identified two RNA binding proteins required for proper neuron-specific splicing. The lab conducted touch- response behavioral assays to assess the function of these proteins in touch-sensing neurons. After isolating C. elegans worms with specific phenotypes, the lab used automated computer tracking and video analysis to record the worms’ behavior. The behavior of mutant worms differed from that of wild-type worms. The Norris lab also discovered two possible RNA binding protein sites in SAD-1, a neuronal gene implicated in the neuronal development of C. elegans1. These two binding sites may control the splicing of SAD-1. The lab transferred mutated DNA into the genome of wild-type worms by injecting a mutated plasmid. The newly transformed worms fluoresced green, indicating that the two binding sites control SAD-1 splicing.

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RNA-binding protein LIN28 is a sensitive marker of ovarian primitive germ cell tumours

Histopathology ◽

10.1111/j.1365-2559.2011.03949.x ◽

2011 ◽

Vol 59 (3) ◽

pp. 452-459 ◽

Cited By ~ 31

Author(s):

Debin Xue ◽

Yan Peng ◽

Fenghua Wang ◽

Robert W Allan ◽

Dengfeng Cao

Keyword(s):

Germ Cell ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Germ Cell Tumours ◽

Sensitive Marker

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Bovine fetal testicular development in the Nellore breed

Semina Ciências Agrárias ◽

10.5433/1679-0359.2017v38n4supl1p2551 ◽

2017 ◽

Vol 38 (4Supl1) ◽

pp. 2551

Author(s):

Juliana Stephany de Souza ◽

Maria Carolina Villani Miguel ◽

Marcos Antônio Maioli ◽

Arthur Nelson Trali Neto ◽

David Giraldo Arana ◽

...

Keyword(s):

Germ Cells ◽

Sertoli Cells ◽

Leydig Cells ◽

Principal Component ◽

Gonadal Development ◽

Cell Number ◽

Testicular Development ◽

Testosterone Concentration ◽

Testicular Weight ◽

Bovine Fetuses

The study of gonadal development improves the understanding of factors that can influence the reproductive development process. This study aims to characterize bovine fetal testicular development and the testosterone level in the Nellore breed. For the study, 162 bovine fetuses aged between 3 and 8 months were collected from Nellore cows at a local abattoir. The fetal age was estimated by DP=8.4+0.087L+5.46?L, where DP is the estimated pregnancy day and L represents fetal length. The fetal gonadal weight (g), width (cm), and thickness (cm) were measured. Thereafter, the gonads were submitted to classic histology processes in 3-µm-thick slices cut at 210 µm intervals. The Sertoli cells, Leydig cells, and germ cells were counted. Blood samples were collected from umbilical cords for testosterone levels. The data were analyzed using the Spearman correlation test followed by Principal Component Analysis and one-way ANOVA to compare the averages between months. The testicular weight and volume were found to have a positive correlation with the numbers of Sertoli cells (r = 0.84; p < 0.0001 and r = 0.92; p < 0.0001, respectively), Leydig cells (r = 0.80; p < 0.0001 and r = 0.90; p < 0.0001, respectively), and germ cells (r = 0.84; p < 0.0001 and r = 0.93; p < 0.0001, respectively) and to be negatively correlated with testosterone plasmatic concentration (r = -0.31; p = 0.0001 and r = -0.22; p = 0.006, respectively) during pregnancy. After the fifth month, the numbers of Sertoli cells, Leydig cells and germ cells differed (p < 0.0001) from the following gestational months. The highest testosterone concentration (p = 0.007) was observed in the fifth month of gestation and was followed by a concentration decrease in the seventh and eighth months. The increase in cell quantity was responsible for the increase in testicular weight and volume during fetal development. On the other hand, the testosterone concentration followed the increase in testicular weight and volume until the 7th month of gestation and regressed during the 8th and 9th months, in addition to the increase in cell number.

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Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis

Reproduction ◽

10.1530/rep-09-0020 ◽

2009 ◽

Vol 138 (4) ◽

pp. 667-677 ◽

Cited By ~ 47

Author(s):

Kyle C Caires ◽

Jeanene de Avila ◽

Derek J McLean

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Germ Cell ◽

Cell Survival ◽

Germ Cells ◽

Pcr Analysis ◽

Vascular Endothelial ◽

Endothelial Growth Factor ◽

Testis Tissue

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

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