Project description:miRNA expression profiles for round spermatids of wild type and GRTH knock-out mice were determined by Rodent TaqMan® Low Density miRNA Arrays A v2.0 (TLDA, Applied Biosystem). Purified round spermatids were prepared from the testis of wild type and GRTH null mice (C57BL/6 strain). Equal amount of total RNA from 20 mice (wild type or GRTH KO) was pooled prior to gene expression analysis.
Project description:miRNA expression profiles for round spermatids of wild type and GRTH knock-out mice were determined by Rodent TaqMan® Low Density miRNA Arrays A v2.0 (TLDA, Applied Biosystem).
Project description:GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility.
Project description:GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility.
Project description:Microarray analysis of purified pachytene spermatocytes and round spermatids. Each stage was examined in wild type and RNF8 knockout mice in two biological replicates.
Project description:Pachytene piRNAs are PIWI-interacting small RNAs abundantly expressed in pachytene spermatocytes and spermatids in adult mouse testes. Both MIWI and MILI-bound pachytene piRNAs have been found enriched in round spermatids. Miwi-null male mice are sterile due to spermiogenic arrest. In C. elegans, sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of offspring derived from piRNA-deficient sperm display a progressive fertility loss after several generations. In mice, it remains unknown whether MIWI-bound pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of offspring derived from MIWI-bound pachytene piRNA-deficient round spermatids also exhibit transgenerational loss of fertility. Here, we report that Miwi KO round spermatids could fertilize both wild type (WT) and Miwi KO oocytes through round spermatid microinjection (ROSI), and produce healthy and fertile offspring despite the aberrant pachytene piRNA profiles in those Miwi KO spermatids. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice. Method: Round spermatids were purified from WT and Miwi KO adult testes using a mini-STA-PUT method[Methods Enzymol 1993; 225:84-113.],the purity of round spermatids was >90% based on our previous report[ J Biol Chem 2012; 287:25173-25190.]. Small RNA was isolated from round spermatids using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were assessed using the Agilent 2100 Bioanalyzer. Small RNA-Seq was performed on an Ion Proton sequencer (Life Technologies). Libraries were prepared using the Ion Total RNA-Seq Kit v2 (Invitrogen) with biological triplicates for WT and Miwi KO samples. Resutls:Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice.
Project description:Microarray analysis of purified pachytene spermatocytes and round spermatids. Each stage was examined in wild type and RNF8 knockout mice in two biological replicates. We performed microarray analysis using Affymetrix Gene 1.0 ST Arrays with purified pachytene spermatocytes and round spermatids. Pachytene spermatocytes and round spermatids were enriched from 3 to 4 males from the WT or Rnf8-KO via BSA gravity sedimentation according to the previous publication [PMID 8231890] and >95% (PS, RS) enrichments were verified after DAPI staining under a fluorescent microscope. For microarray analysis, total RNAs from purified pachytene spermatocytes or round spermatids were examined.
Project description:The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO, underscore the relevance of GRTH in translation. Studies showed the association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. Microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice.
Project description:Actin-related proteins (Arp) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp and highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, while heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects such as detached acrosomes, disrupted membranes and failed elongation of the axoneme start to appear at spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids, were degraded. Co-immunoprecipitation experiments identified interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establishes that mutations in ACTL7B are directly related to male infertility, pressing for additional research in men.