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

TATA-binding protein (TBP)-associated factor 7l (Taf7l; a paralogue of Taf7) and TBP-related factor 2 (Trf2) are components of the core promoter complex required for gene/tissue-specific transcription of protein-coding genes by RNA polymerase II. Previous studies reported that Taf7l knockout (KO) mice exhibit structurally abnormal sperm, reduced sperm count, weakened motility, and compromised fertility. Here we find that continued backcrossing of Taf7l(-/Y) mice from N5 to N9 produced KO males that are essentially sterile. Genome-wide expression profiling by mRNA-sequencing analysis of wild-type (WT) and Taf7l(-/Y) (KO) testes revealed that Taf7l ablation impairs the expression of many postmeiotic spermatogenic-specific as well as metabolic genes. Importantly, histological analysis of testes revealed that Taf7l(-/Y) mice develop postmeiotic arrest at the first stage of spermiogenesis, phenotypically similar to Trf2(-/-) mice, but distinct from Taf4b(-/-) mice. Indeed, we find that Taf7l and Trf2 coregulate postmeiotic genes, but none of Taf4b-regulated germ stem cell genes in testes. Genome-wide ChIP-sequencing studies indicate that TAF7L binds to promoters of activated postmeiotic genes in testis. Moreover, biochemical studies show that TAF7L associates with TRF2 both in vitro and in testis, suggesting that TAF7L likely cooperates directly with TRF2 at promoters of a subset of postmeiotic genes to regulate spermiogenesis. Our findings thus provide a previously undescribed mechanism for cell-type-specific transcriptional control involving an interaction between a "nonprototypic" core promoter recognition factor (Trf2) and an orphan TAF subunit (Taf7l) in mammalian testis-specific gene transcription. 

Taf7l cooperates with Trf2 to regulate spermiogenesis.

Piwi-piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5'-to-3' directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing. 

The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.

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.

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

Liver serine/threonine kinase B1 (LKB1) is a tumor suppressor associated with the pathogenesis of Peutz-Jeghers syndrome. Affected males are at increased risk of developing Sertoli cell tumors and display defective spermatogenesis. Male mice lacking the short isoform (Lkb1) of Lkb1 were sterile and exhibited abnormal spermiogenesis. In addition to the short isoform, the long isoform of Lkb1 (Lkb1) is also expressed in testis; however, the requirement of the long isoform for fertility and the functional difference between the isoforms remain unknown. Herein, different from the spermiation failure reported in Lkb1 knockout mice, conditional deletion (cKO) of both isoforms of Lkb1 in germ cells resulted in male sterility stemming from defects in acrosome formation, as well as nuclear elongation and condensation during spermatid differentiation. Additionally, cKO mice showed a progressive germ cell loss that was never reported in mice with Lkb1 deletion. Further experiments revealed that the defect resulted from the failure of spermatogonial stem/progenitor cells (SPCs) maintenance. Although increased mTORC1 activity in postnatal cKO testes was consistent with a tendency toward germline stem cell differentiation, in vivo inhibition of the pathway by rapamycin treatment failed to rescue the phenotype. Concurrently, we detected a significant reduction of mitochondrial activity in Lkb1deficient SPCs. The results suggest that the regulation of LKB1 on SPCs' maintenance is associated with mitochondrial functions but not through the mTOR signaling pathway. In summary, our study supports different roles of Lkb1 isoforms in spermatogenesis with Lkb1 directing SPCs maintenance, and Lkb1 and Lkb1 coordinately regulating spermatid differentiation.

Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis.

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.

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.

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.

Biological and RNA regulatory function of MOV10 in mammalian germ cells.

MOV10L1 and its paralog MOV10 are evolutionally conserved RNA helicases involved in distinct RNA regulatory pathways. The testis-specific MOV10L1 is essential for spermatogenesis and PIWI-interacting RNAs biogenesis, whereas MOV10 is ubiquitous and multifunctional. Although both proteins have been implied to correlate with RNA G-quadruplex (RG4) in vivo, their capabilities in binding and resolving RG4 and their respective biological significance remain unclear. Herein, we comprehensively characterize and compare the activities of these two helicases on various nucleic acid substrates in vitro, with a focus on RG4 structure. We find that both MOV10L1 and MOV10 are able to resolve RG4, with MOV10L1 being more efficient in that. In contrast to MOV10, MOV10L1 prefers to bind to a junction between single-stranded RNA and RG4, which is mediated by both its N and C termini. Furthermore, we show that RG4 unwinding by MOV10L1 facilitates the cleavage of this specific RNA structure by an endonuclease.

MOV10L1 Binds RNA G-Quadruplex in a Structure-Specific Manner and Resolves It More Efficiently Than MOV10.

Although it is widely believed that China is facing a major shortage of pediatricians, the real situation of the current national status of pediatric human resources and their working conditions has not been evaluated to date.

ArticleTotal : 3	Year
Publication title Cited by 6	2018
Publication title Cited by 13	2019
Publication title Cited by 10	2019

Article	Year
Taf7l cooperates with Trf2 to regulate spermiogenesis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24082143	2013
The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing. Genes & development\| PubMed ID: 25762440	2015
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
An essential role for PNLDC1 in piRNA 3' end trimming and male fertility in mice. Cell research\| PubMed ID: 28994417	2017
Differential regulation of spermatogenic process by Lkb1 isoforms in mouse testis. Cell death & disease\| PubMed ID: 29022902	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
Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis. Aging\| PubMed ID: 30636722	2019
Biological and RNA regulatory function of MOV10 in mammalian germ cells. BMC biology\| PubMed ID: 31088452	2019
MOV10L1 Binds RNA G-Quadruplex in a Structure-Specific Manner and Resolves It More Efficiently Than MOV10. iScience\| PubMed ID: 31252377	2019
Characteristics and Workload of Pediatricians in China. Pediatrics\| PubMed ID: 31253739	2019

Ke Zheng

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