an-in-vivo-method-to-study-mouse-blood-testis-barrier-integrity

An <em>In Vivo</em> Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

enhanced-crosslinking-immunoprecipitation-eclip-method-for-efficient

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, ...

in-vitro-biochemical-assays-using-biotin-labels-to-study-protein

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Nanjing Medical University

Rapamycin extends lifespan in mice, but can have a number of undesirable effects that may ultimately limit its utility in humans. The canonical target of rapamycin, and the one thought to account for its effects on lifespan, is the mammalian/mechanistic target of rapamycin, complex 1 (mTORC1). We have previously shown that at least some of the detrimental side effects of rapamycin are due to "off target" disruption of mTORC2, suggesting they could be avoided by more specific targeting of mTORC1. However, mTORC1 inhibitionper se can reduce the mRNA expression of mitochondrial genes and compromise the function of mitochondria in cultured muscle cells, implying that defects in bioenergetics might be an unavoidable consequence of targeting mTORC1 in vivo. Therefore, we tested whether rapamycin, at the same doses used to extend lifespan, affects mitochondrial function in skeletal muscle. While mitochondrial transcripts were decreased, particularly in the highly oxidative soleus muscle, we found no consistent change in mitochondrial DNA or protein levels. In agreement with the lack of change in mitochondrial components, rapamycin-treated mice had endurance equivalent to that of untreated controls, and isolated, permeabilized muscle fibers displayed similar rates of oxygen consumption. We conclude that the doses of rapamycin required to extend life do not cause overt mitochondrial dysfunction in skeletal muscle.

Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance.

Rapamycin extends life span in mice, yet paradoxically causes lipid dysregulation and glucose intolerance through mechanisms that remain incompletely understood. Whole-body energy balance can be influenced by beige/brite adipocytes, which are inducible by cold and other stimuli via β-adrenergic signaling in white adipose depots. Induction of beige adipocytes is considered a promising strategy to combat obesity because of their ability to metabolize glucose and lipids, dissipating the resulting energy as heat through uncoupling protein 1. Here, we report that rapamycin blocks the ability of β-adrenergic signaling to induce beige adipocytes and expression of thermogenic genes in white adipose depots. Rapamycin enhanced transcriptional negative feedback on the β3-adrenergic receptor. However, thermogenic gene expression remained impaired even when the receptor was bypassed with a cell-permeable cAMP analog, revealing the existence of a second inhibitory mechanism. Accordingly, rapamycin-treated mice are cold intolerant, failing to maintain body temperature and weight when shifted to 4°C. Adipocyte-specific deletion of the mTORC1 subunit Raptor recapitulated the block in β-adrenergic signaling. Our findings demonstrate a positive role for mTORC1 in the recruitment of beige adipocytes and suggest that inhibition of β-adrenergic signaling by rapamycin may contribute to its physiological effects.

Rapamycin Blocks Induction of the Thermogenic Program in White Adipose Tissue.

Cytokine-dependent renewal of stem cells is a fundamental requisite for tissue homeostasis and regeneration. Spermatogonial progenitor cells (SPCs) including stem cells support life-long spermatogenesis and male fertility, but pivotal phosphorylation events that regulate fate decisions in SPCs remain unresolved. Here, we described a quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of SPCs following sustained stimulation with glial cell-derived neurotrophic factor (GDNF), an extrinsic factor supporting SPC proliferation. Stimulated SPCs contained 3382 identified phosphorylated proteins and 12141 phosphorylation sites. Of them, 325 differentially phosphorylated proteins and 570 phosphorylation sites triggered by GDNF were highly enriched for ERK1/2, GSK3, CDK1, and CDK5 phosphorylating motifs. We validated that inhibition of GDNF/ERK1/2-signaling impaired SPC proliferation and increased G2/M cell cycle arrest. Significantly, we found that proliferation of SPCs requires phosphorylation of the mTORC1 component Raptor at Ser Tissue-specific deletion of  in mouse germline cells results in impaired spermatogenesis and progressive loss of spermatogonia, but  increased phosphorylation of Raptor by raptor over-expression in SPCs induced a more rapidly growth of SPCs in culture. These findings implicate previously undescribed signaling networks in governing fate decision of SPCs, which is essential for the understanding of spermatogenesis and of potential consequences of pathogenic insult for male infertility.

The Glial Cell-Derived Neurotrophic Factor (GDNF)-responsive Phosphoprotein Landscape Identifies Raptor Phosphorylation Required for Spermatogonial Progenitor Cell Proliferation.

Rapamycin is a clinically important drug that is used in transplantation and cancer therapy but which causes a number of side effects, including male infertility. Its canonical target, mammalian target of rapamycin complex 1 (mTORC1), plays a key role in metabolism and binds chromatin; however, its precise role in the male germline has not been elucidated. Here, we inactivate the core component, Raptor, to show that mTORC1 function is critical for male meiosis and the inactivation of sex chromosomes. Disruption of the  gene impairs chromosomal synapsis and prevents the efficient spreading of silencing factors into the XY chromatin. Accordingly, mRNA for XY-linked genes remains inappropriately expressed in -deficient mice. Molecularly, the failure to suppress gene expression corresponded with deficiencies in 2 repressive chromatin markers, H3K9 dimethylation and H3K9 trimethylation, in the XY body. Together, these results demonstrate that mTORC1 has an essential role in the meiotic progression and silencing of sex chromosomes in the male germline, which may explain the infertility that has been associated with such inhibitors as rapamycin.-Xiong, M., Zhu, Z., Tian, S., Zhu, R., Bai, S., Fu, K., Davis, J. G., Sun, Z., Baur, J. A., Zheng, K., Ye, L. Conditional ablation of  in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes.

Conditional ablation of  in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes.

CYP19 is the single copy gene encoding for the estrogen synthetic enzyme aromatase. Alternate splicing of the promoter is the regulatory mechanism of this gene. In the brain, estrogen is synthesized in neuronal and glial cells and the gene is mainly regulated by the alternate promoter PI.f. The hormone produced in this vicinity has been associated with maintaining normal brain functions. Previously, epigenetic regulation has been shown in the promoters PII and I.3 of CYP19 in adipocytes. In the present study, the methylation of PI.f in CYP19 was examined in glial cells. Treatment of the hypomethylating agent 5-aza-2'deoxycytidine increased CYP19 mRNA species in U87 MG cells while little changes were observed in the other glia cell lines. As PI.f is also chiefly used in T98G cells with high expression of CYP19, the methylation statuses of the promoter in these two cell models were compared. Our results showed that treating U87 MG cells with 10 μM 5-aza-2'deoxycytidine significantly induced a ∼10-fold increase in CYP19 transcription and ∼80% increase in aromatase activity. In contrast, the same treatment did not change either endpoint in T98G cells. Further investigation illustrated the CpGs in PI.f were differentially methylated in the two cell lines; 63% and 37% of the 14 CpG sites were methylated in U87 MG and T98G cells respectively. In conclusion, this study illustrated that the brain-specific PI.f derived CYP19 expression can be regulated by DNA methylation.

Methylation dictates PI.f-specific CYP19 transcription in human glial cells.

An essential role for PNLDC1 in piRNA 3' end trimming and male fertility in mice.

What is the physiological role of Rictor in spermatogenic cells?

A germline-specific role for the mTORC2 component Rictor in maintaining spermatogonial differentiation and intercellular adhesion in mouse testis.

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids.  mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most  mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including , ,  and  These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.

Long non-coding RNAs (lncRNA) are emerging as new genetic/epigenetic regulators that can impact almost all physiological functions. Here, we focus on the long non-coding steroid receptor RNA activator (SRA), including new insights into its effects on gene expression, the cell cycle, and differentiation; how these relate to physiology and disease; and the mechanisms underlying these effects. We discuss how SRA acts as an RNA coactivator in nuclear receptor signaling; its effects on steroidogenesis, adipogenesis, and myocyte differentiation; the impact on breast and prostate cancer tumorigenesis; and, finally, its ability to modulate hepatic steatosis through several signaling pathways. Genome-wide analysis reveals that SRA regulates hundreds of target genes in adipocytes and breast cancer cells and binds to thousands of genomic sites in human pluripotent stem cells. Recent studies indicate that SRA acts as a molecular scaffold and forms networks with numerous coregulators and chromatin-modifying regulators in both activating and repressive complexes. We discuss how modifications to SRA's unique stem-loop secondary structure are important for SRA function, and highlight the various SRA isoforms and mutations that have clinical implications. Finally, we discuss the future directions for better understanding the molecular mechanisms of SRA action and how this might lead to new diagnostic and therapeutic approaches.

New Insights Into the Long Non-coding RNA SRA: Physiological Functions and Mechanisms of Action.

Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis.

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.

Dual functions for the ssDNA-binding protein RPA in meiotic recombination.

RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells.

ArticleTotal : 3	Year
Publication title	2018
Publication title	2019
Publication title	2019

Article	Year
Rapamycin doses sufficient to extend lifespan do not compromise muscle mitochondrial content or endurance. Aging\| PubMed ID: 23929887	2013
Rapamycin Blocks Induction of the Thermogenic Program in White Adipose Tissue. Diabetes\| PubMed ID: 26858361	2016
The Glial Cell-Derived Neurotrophic Factor (GDNF)-responsive Phosphoprotein Landscape Identifies Raptor Phosphorylation Required for Spermatogonial Progenitor Cell Proliferation. Molecular & cellular proteomics : MCP\| PubMed ID: 28408662	2017
Conditional ablation of in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 28490482	2017
Methylation dictates PI.f-specific CYP19 transcription in human glial cells. Molecular and cellular endocrinology\| PubMed ID: 28559115	2017
An essential role for PNLDC1 in piRNA 3' end trimming and male fertility in mice. Cell research\| PubMed ID: 28994417	2017
A germline-specific role for the mTORC2 component Rictor in maintaining spermatogonial differentiation and intercellular adhesion in mouse testis. Molecular human reproduction\| PubMed ID: 29518209	2018
Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes. Development (Cambridge, England)\| PubMed ID: 29866902	2018
New Insights Into the Long Non-coding RNA SRA: Physiological Functions and Mechanisms of Action. Frontiers in medicine\| PubMed ID: 30238005	2018
Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis. Aging\| PubMed ID: 30636722	2019
Dual functions for the ssDNA-binding protein RPA in meiotic recombination. PLoS genetics\| PubMed ID: 30716097	2019
Biological and RNA regulatory function of MOV10 in mammalian germ cells. BMC biology\| PubMed ID: 31088452	2019

Lan Ye

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