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ShRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

ABSTRACT: RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA – Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE64324 | GEO | 2014/12/19

SECONDARY ACCESSION(S): PRJNA270755

REPOSITORIES: GEO

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shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

Project description:RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA – Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo. Comparison of total RNA from post-natal day 15 testis extracted from three wild-type mice and three transgenic mice genetically engineered to express siRNA designed to knock-down Rhox3. RNA hybridised to Affymetrix mouse all exon arrays.

2014-12-19 | E-GEOD-64324 | biostudies-arrayexpress

KDM6A/UTX promotes spermatogenic gene expression across generations but is dispensable for male fertility

Project description:Paternal chromatin undergoes extensive structural and epigenetic changes during mammalian spermatogenesis, producing sperm with an epigenome optimized for the transition to embryogenesis. Lysine demethylase 6a (KDM6A, also called UTX) promotes gene activation in part via demethylation of H3K27me3, a developmentally important repressive modification abundant throughout the epigenome of spermatogenic cells and sperm. We previously demonstrated increased cancer risk in genetically wild type mice derived from a paternal germ line lacking Kdm6a (Kdm6a cKO), indicating a role for KDM6A in regulating heritable epigenetic states. However, the regulatory function of KDM6A during spermatogenesis is not known. Here, we show that Kdm6a is transiently expressed in spermatogenesis, with RNA and protein expression largely limited to late spermatogonia and early meiotic prophase. Kdm6a cKO males do not have defects in fertility or the overall progression of spermatogenesis. However, hundreds of genes are deregulated upon loss of Kdm6a in spermatogenic cells, with a strong bias towards downregulation coinciding with the time when Kdm6a is expressed. Misregulated genes encode factors involved in chromatin organization and regulation of repetitive elements, and a subset of these genes was persistently deregulated in the male germ line across two generations of offspring of Kdm6a cKO males. Genome-wide epigenetic profiling revealed broadening of H3K27me3 peaks in differentiating spermatogonia of Kdm6a cKO mice, suggesting that KDM6A demarcates H3K27me3 domains in the male germ line. Our findings highlight KDM6A as a transcriptional activator in the mammalian male germ line that is dispensable for spermatogenesis but important for safeguarding gene regulatory state intergenerationally.

2023-10-24 | GSE215112 | GEO

Proteome and phosphoproteome of Wip1-knockout mouse testis

Project description:Spermatogenesis plays an important role in the mammalian testis, involving in the complex processes of mitosis, meiosis, and spermiogenesis. Spermatogenesis may also be disrupted in the absence of the immunological and ‘fence’ functions of the BTB, resulting in male subfertility or infertility. Mice lacking wild-type p53-induced phosphatase 1 (Wip1) display male reproductive organ defects, but the molecular mechanisms underlying these abnormalities remain unclear. We explored the function of Wip1 in spermatogenesis and fertility by examining differences in the expressed testis proteome and phosphoproteome between Wip1-deficient and wild-type mice using a proteomics approach. 90 proteins and 178 phosphoproteins were differentially regulated between these two groups of mice. These results suggested that proinflammatory cytokines may impair the blood–testis barrier dynamics by decreasing the expression of junction-associated proteins, which effect could be partially responsible for the subfertility and spermatogenesis defects in Wip1-knockout mice.

2022-03-01 | PXD008277 | Pride

Integrative proteomic and phosphoproteomic profiling of testis from Wip1-knockout mice: insights into mechanisms of reduced fertility

Project description:Spermatogenesis plays an important role in the mammalian testis, involving in the complex processes of mitosis, meiosis, and spermiogenesis. Spermatogenesis may also be disrupted in the absence of the immunological and ‘fence’ functions of the BTB, resulting in male subfertility or infertility. Mice lacking wild-type p53-induced phosphatase 1 (Wip1) display male reproductive organ defects, but the molecular mechanisms underlying these abnormalities remain unclear. We explored the function of Wip1 in spermatogenesis and fertility by examining differences in the expressed testis proteome and phosphoproteome between Wip1-deficient and wild-type mice using a proteomics approach. Among a total of 6872 proteins and 4280 phosphorylation sites on 1614 proteins identified in our analysis, 58 proteins and 159 phosphorylation sites on 141 proteins were differentially regulated between these two groups of mice. These results suggested that proinflammatory cytokines may impair the blood–testis barrier dynamics by decreasing the expression of junction-associated proteins, which effect could be partially responsible for the subfertility and spermatogenesis defects in Wip1-knockout mice.

2018-11-02 | PXD009220 | Pride

Expression profiling of mouse ing2 -/- mice with spermatogenic arrest and infertility

Project description:Expression profiling of mouse ing2 -/- testis vs WT reveals gene expression differences consistent with spermatogenic arrest and infertility. Ing2 is indispensable for male germ cell development in mice. While mice deficient for Ing2 were born and grew without apparent abnormalities, male, but not female, were infertile, consistent with the highest expression of Ing2 in testes in wild-type mice and in humans. Histological and DNA content analyses in Ing2-/- testes revealed a spermatogenesis arrest at meiotic phase and enhanced apoptosis associated with increased p53, resulting in a decline in mature spermatozoa, which became more severe in older age. HDAC1 accumulation and core histone deacetylation at pachytene stage were impaired in Ing2-/- testes, suggesting that the recruitment of HDAC1 by Ing2 plays a critical role in spermatogenesis. This study establishes Ing2 as a novel mammalian regulator of spermatocyte differentiation, which coordinates spermatogenesis stage-specific histone modifications. This study has implications in understanding human male infertility. Testis from WT and Ing2 KO mice at 2-3 months of age where analyzed for differences in gene expression.

2010-11-19 | E-GEOD-18610 | biostudies-arrayexpress

Methylome study on human spermatogenic cells

Project description:DNA methylation is an important regulator of the chromatin structure and is necessary for gene expression stability and maintenance of cell identitiy. However, to which extent DNA methylation changes during the key differentiation steps of human spermatogenesis remains largely unkown. Here, we generated genome wide methylomes (Enzymatic Methyl-sequencing) of human undifferentiated spermatogonia (Undiff), differentiating spermatogonia (Diff), tetraploid spermatocytes (4C) and haploid spermatids (1C) obtained from testicular tissues of three patients with qualitatively and quantitatively normal spermatogenesis (control, CTR). We also generated Undiff, Diff and 4C methylomes from two patients with impaired spermatogenesis (cryptozoospermic, CZ). Our study is the first to report about DNA methylation changes during human spermatogenesis.

| EGAS00001007449 | EGA

Expression profiling of mouse ing2 -/- mice with spermatogenic arrest and infertility

2010-11-19 | GSE18610 | GEO

Microarray analysis of human spermatogenic dysfunction

Project description:We investigated testicular biopsies from 28 men which presented with full spermatogenesis (Johnsen Score 10, 12 samples), arrest at the spermatid stage (Johnsen Score 8, 6 samples), arrest at spermatocyte stage (Johnsen Score 5, 5 samples) and Sertoli-cell-only syndrom (Johnsen Score 2, 5 samples). Testicular biopsies were obtained by the "sandwich" method and conserved in RNALater (Ambion) during surgery. Keywords: spermatogenesis germ cell differentiation Keywords: disease state analysis

2006-05-12 | GSE4797 | GEO

Proper chromatin programming during spermatogenesis is required for embryonic development

Project description:Epigenetic mechanisms including DNA methylation, non-coding RNAs and histone modifications control gene expression. Studies suggest that a father's lifetime experiences can be transmitted to his offspring to affect development and health. The mechanisms underlying such epigenetic inheritance are unknown. A potential route for paternal transmission is the unique chromatin composition of spermatozoa. Unlike somatic cells and oocytes, most nucleosomes in sperm are replaced with protamine nucleoproteins. The role of residual nucleosomes, residing at gene regulatory sequences, for epigenetic control of embryonic development is unknown. Here we generated a transgenic mouse model in which over-expression of the histone H3 lysine 4 (H3K4) demethylase LSD1/KDM1A during spermatogenesis alters H3K4 methylation in sperm. Strikingly, KDM1A over-expression in one generation causes severe embryonic defects in non-transgenic descendants spanning three subsequent generations. We show for the first time that correct histone methylation homeostasis during spermatogenesis is critical for offspring development and survival over multiple generations. Identification of H3K4me2 and nucleosome occupancies in sperm of wildtype mice, KDM1A transgenic mice and their non-transgenic littermates.

2015-11-03 | E-GEOD-55471 | biostudies-arrayexpress

Microarray profiling of different spermatogenic cells

Project description:To understand the changes in gene expression during spermatogenesis, we did microarray profiling of spermatogonial stem cells, leptotene-zygotene cells and round spermatids. Microarray profiling of pachytene-diplotene cells can be found in E-MTAB-2668.

2019-01-14 | E-MTAB-7067 | biostudies-arrayexpress

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