microRNA-383 impairs phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest in testicular embryonal carcinoma cells

Male germ cells with aberrant DNA damage are the weighted factor contributing to male infertility. Mounting evidence shows that DNA damage in male germ cells impairs spermatogenesis and lowers fecundity. MicroRNAs (miRNAs) regulating expression of multiple genes play a significant role in spermatogenesis. Our previous results have shown that microRNA-383 (miR-383) is one of the notable down-regulated microRNAs in the testes of sterile males with maturation arrest (MA) and is located predominantly in spermatogonia and primary spermatocytes. However, the role that miR-383 plays in DNA damage during spermatogenesis remains unknown. In this study, we found that miR-383 inhibited the focal formation and abundance of γH2AX, which is the major marker of sites of DNA damage, with or without ultraviolet irradiation and cisplatin in testicular embryonal carcinoma (NT-2) cells. In addition, NT-2 cells were remarkably sensitized to DNA damage reagent (cisplatin) by forcing expression of miR-383 and silencing expression of protein phosphatase 1, regulatory subunit 10 (PNUTS). By constructing Renilla luciferase reporters and co-transfecting miR-383 and reporters in NT-2 cells, we identified that PNUTS was a valid target of miR-383. Further results demonstrated that the repression of the phosphorylated form of H2AX by miR-383 was due to independent depletion of PNUTS and cell cycle arrest. In conclusion, we found a novel function of miR-383 in the DNA damage pathway. miR-383 impairs the phosphorylation of H2AX by targeting PNUTS and inducing cell cycle arrest independently, as well as sensitizing NT-2 cells to cisplatin.     

Three Drosophila Hox complex microRNAs do not have major effects on expression of evolutionarily conserved Hox gene targets during embryogenesis

The discovery of microRNAs has resulted in a major expansion of the number of molecules known to be involved in gene regulation. Elucidating the functions of animal microRNAs has posed a significant challenge as their target interactions with messenger RNAs do not adhere to simple rules. Of the thousands of known animal microRNAs, relatively few microRNA:messenger RNA regulatory interactions have been biologically validated in an normal organismal context. Here we present evidence that three microRNAs from the Hox complex in Drosophila (miR-10-5p, miR-10-3p, miR-iab-4-5p) do not have significant effects during embryogenesis on the expression of Hox genes that contain high confidence microRNAs target sites in the 3' untranslated regions of their messenger RNAs. This is significant, in that it suggests that many predicted microRNA-target interactions may not be biologically relevant, or that the outcomes of these interactions may be so subtle that mutants may only show phenotypes in specific contexts, such as in environmental stress conditions, or in combinations with other microRNA mutations.     

miR-7 and miR-218 epigenetically control tumor suppressor genes RASSF1A and Claudin-6 by targeting HoxB3 in breast cancer

Many microRNAs have been implicated as key regulators of cellular growth and differentiation and have been found to dysregulate proliferation in human tumors, including breast cancer. Cancer-linked microRNAs also alter the epigenetic landscape by way of DNA methylation and post-translational modifications of histones. Aberrations in Hox gene expression are important for oncogene or tumor suppressor during abnormal development and malignancy. Although recent studies suggest that HoxB3 is critical in breast cancer, the putative role(s) of microRNAs impinging on HoxB3 is not yet fully understood. In this study, we found that the expression levels of miR-7 and miR-218 were strongly and reversely associated with HoxB3 expression. Stable overexpression of miR-7 and miR-218 was accompanied by reactivation of tumor suppressor genes including RASSF1A and Claudin-6 by means of epigenetic switches in DNA methylation and histone modification, giving rise to inhibition of the cell cycle and clone formation of breast cancer cells. The current study provides a novel link between overexpression of collinear Hox genes and multiple microRNAs in human breast malignancy.     

Mouse hybrid sterility and testicular function

Crosses of BALB/c female mice and inbred wild male mice (PWD, PWK) produce fertile female progeny, but the male offspring are sterile. The hybrid male sterility is a direct action of the hybrid sterility genes Hst-1s and Hstws. Previous reports concluded that spermatogenic arrest effected the sterility. However, the testicular steroidogenesis of hybrid sterile male mice has not been elucidated. In the present report, the steroidogenic capacity of hybrid sterile and parental strain males was directly assessed by quantifying testosterone secretion by maximally stimulated testes perfused in vitro. Additionally, Leydig cell mass and germ cell volumes were morphometrically determined. The experimental results confirm the deleterious impact of the Hst-1s/Hstws genotype on spermatogenesis and demonstrate for the first time that the steroidogenic capacity of hybrid sterile testes is reduced. The biochemical defects that cause the impairment of testicular function are unknown.     

Male germ cells require polyenoic sphingolipids with complex glycosylation for completion of meiosis: a link to ceramide synthase-3

Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26-C32) polyunsaturated (4-6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1(-/-) mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.     

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.     

The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.     

Anomalies de position des testicules dans l’enfance: conséquences à l’âge adulte

Non descended testes in the low scrotum is a common anomaly at birth, with about 4% of the newborn males affected. Only one quarter of these newborn babies will still have non descended testes when one year old. However, the testes that will descend within the first year of life seem no more to be considered as normally descended testes. Moreover, the retractile testis, which represents a secondary anomaly of testicular position occuring after the babies are older than one year, is no more to be regarded as a physiological variant of the normally descended testis, since several reports indicate histological and clinical modifications in such cases. The testicular non descent can be associated with two consequences in adult life. Firstly, an history of non descended testis is the only known risk factor for the testicular cancer. Secondly, such an history is a risk factor for the male fertility because of spermatogenesis alterations, as indicated by qualitative and quantitative histological analyses of the testicular tissue, and by depressed spermatozoa output and quality (motility, normal forms); moreover, testicular volumes are reduced, and the time to pregnancy as well as the rate of infertility are increased. Time is arrived for a reappraisal of the consequences in adult life of the abnormal testicular location (either congenital or acquired) during childhood.     

The effect of xenobiotics on microRNA expression in the rat liver

The effect of xenobiotics on microRNA expression in the rat liver has been investigated. Based on results of bioinformatics analysis several microRNAs that can interact with 3'-untranslated regions of cytochrome P450 (CYP) mRNAs have been selected. These included three microRNAs (miR-21, miR-221, miR-222) for CYP1A1 mRNA as a putative target and two microRNAs (miR-143, miR-152) for CYP2B1 mRNA as a putative target. Using the RT-PCR method, expression levels of these microRNAs have been detected in the liver of rats treated with inducers of CYP1A and CYP2B, benzo(a)pyrene (BP), phenobarbital (PB), and DDT. In rats treated with both BP and DDT the hepatic content of miR-21, miR-221 and miR-222 was 2?3 times lower than in the control animals, while ethoxyresorufin-O-deethylase (EROD) activity of CYP1A1 demonstrated a 5.5?8.7-fold increase. In PB-treated rats miR-143 expression remained unchanged, the level of miR-152 increased 2-fold, while pentoxyresorufin-O-deetylase (PROD) activity of CYP2B increased 10.5-fold. In the liver of DDT-treated rats PROD activity demonstrated a 20.8-fold increase; expression of miR-143 increased 2-fold, and miR-152 expression remained unchanged. Bioinformatics analysis of putative miR-target interactions showed that the selected microRNAs can potentially bind such target as AhR, ESR1, GR, CCND1, PTEN mRNAs. Thus, the expression profile of miR-21, miR-221, miR-222, miR-143, miR-152 may vary in dependence on the CYP inducer used. Analysis in silico has shown that besides genes encoding CYP1A/2B other genes including those involved in hormonal carcinogenesis should be considered as potential targets of the investigated microRNAs.     

KNOCKDOWN OF ATPsyn‐b CAUSED LARVAL GROWTH DEFECT AND MALE INFERTILITY IN Drosophila

The ATPsyn‐b encoding for subunit b of ATP synthase in Drosophila melanogaster is proposed to act in ATP synthesis and phagocytosis, and has been identified as one of the sperm proteins in both Drosophila and mammals. At present, its details of functions in animal growth and spermatogenesis have not been reported. In this study, we knocked down ATPsyn‐b using Drosophila lines expressing inducible hairpin RNAi constructs and Gal4 drivers. Ubiquitous knockdown of ATPsyn‐b resulted in growth defects in larval stage as the larvae did not grow bigger than the size of normal second‐instar larvae. Knockdown in testes did not interrupt the developmental excursion to viable adult flies, however, these male adults were sterile. Analyses of testes revealed disrupted nuclear bundles during spermatogenesis and abnormal shaping in spermatid elongation. There were no mature sperm in the seminal vesicle of ATPsyn‐b knockdown male testes. These findings suggest us that ATPsyn‐b acts in growth and male fertility of Drosophila.     

Testis-derived microRNA profiles of African clawed frogs (Xenopus) and their sterile hybrids

Gene regulation was long predicted to play a vital role in speciation and species divergence. Only recently with the advent of new technologies, however, has it been possible to address the question of the relative contributions of different mechanisms of gene expression to the evolution of phenotypic diversity. Here we broaden the question and ask whether microRNAs, a large class of small regulatory RNAs, play a role in reproductive isolation between species by contributing to hybrid male sterility. MicroRNAs from the testes of clawed frogs (Xenopus) were extracted and the expression profiles of sterile hybrids were compared with males of a parental species. Hybrid testes were largely microRNA-depleted relative to those of nonhybrids, and this pattern was validated with quantitative RT-PCR. A number of candidate differential microRNAs from this study have previously been described as testis-specific in the mouse, suggesting that microRNA structural conservation may be associated with functional retention.     

翘嘴鳜miR-222的表达特征

为了解翘嘴鳜miR-222的时空表达规律, 研究利用实时荧光定量PCR的方法检测miR-222在翘嘴鳜不同组织、胚胎发育及胚后发育中的相对表达丰度。研究结果显示, miR-222在肌肉相关的组织中表达较高, 特别是在成年翘嘴鳜的白肌中表达最高; 胚胎发育阶段结果显示, miR-222在胚胎发育的2细胞期就有表达, 而表达量在心动期达到最高。不同组织及不同发育阶段的差异性表达结果表明, miR-222很可能参与调控鳜鱼肌肉的生长发育。为研究合成代谢过程中miR-222在肌肉生长调控中的表达规律, 通过对翘嘴鳜幼鱼在饥饿一周后饱食一餐的实验处理下, 利用实时荧光定量的方法测定miR-222在骨骼肌中的相对表达变化。结果显示, miR-222的表达量在恢复喂食后的1h显著上升(P0.05), 表明miR-222很可能是调节鱼类骨骼肌生长过程中, 参与快速应答信号系统的一类miRNA。研究为miR-222在鱼类发育中的调控作用提供一些理论依据。       

The Drosophila fragile X-related gene regulates axoneme differentiation during spermatogenesis

Macroorchidism (i.e., enlarged testicles) and mental retardation are the two hallmark symptoms of Fragile X syndrome (FraX). The disease is caused by loss of fragile X mental retardation protein (FMRP), an RNA-binding translational regulator. We previously established a FraX model in Drosophila, showing that the fly FMRP homologue, dFXR, acts as a negative translational regulator of microtubule-associated Futsch to control stability of the microtubule cytoskeleton during nervous system development. Here, we investigate dFXR function in the testes. Male dfxr null mutants have the enlarged testes characteristic of the disease and are nearly sterile (>90% reduced male fecundity). dFXR protein is highly enriched in Drosophila testes, particularly in spermatogenic cells during the early stages of spermatogenesis. Cytological analyses reveal that spermatogenesis is arrested specifically in late-stage spermatid differentiation following individualization. Ultrastructurally, dfxr mutants lose specifically the central pair microtubules in the sperm tail axoneme. The frequency of central pair microtubule loss becomes progressively greater as spermatogenesis progresses, suggesting that dFXR regulates microtubule stability. Proteomic analyses reveal that chaperones Hsp60B-, Hsp68-, Hsp90-related protein TRAP1, and other proteins have altered expression in dfxr mutant testes. Taken together with our previous nervous system results, these data suggest a common model in which dFXR regulates microtubule stability in both synaptogenesis in the nervous system and spermatogenesis in the testes. The characterization of dfxr function in the testes paves the way to genetic screens for modifiers of dfxr-induced male sterility, as a means to efficiently dissect FMRP-mediated mechanisms.