Project description:Testes were collected from 16-day-old wild-type and Hsf5 knockout mice; small RNA libraries were constructed and sequenced on the Illumina NovaSeq 6000 platform, with adapter trimming, extraction of 18–32 nt reads for piRNA analysis, alignment to miRBase, non-coding RNAs, piRNA clusters, and transposable elements (RepeatMasker, mm39), categorization of aligned 25–32 nt piRNAs, and ping-pong analysis using LINE1 and IAP consensus sequences from Repbase, to determine whether HSF5 deficiency affects piRNA biogenesis and transposon silencing during mouse spermatogenesis by assessing piRNA abundance, length distribution, annotation profile, and ping-pong amplification in Hsf5-/- testes
Project description:We used H3f3b-/- testes to analyze the impact of H3f3b derived H3.3 on spermatogenesis. Analysis of Histone H3 (Lys 4) tri-methylation in H3f3b WT/WT and H3f3b -/- testes tissue.
Project description:The disinhibition of transposon elements (TEs) is a significant threat to male reproduction, particularly during the delicate process of spermatogenesis. Here we identify that Death associated protein 6 (Daxx), a potent transcription repressor and an H3.3 chaperone essential for the TEs silencing during spermatogenesis. DAXX-deficient mouse display delayed meiotic progression, reduced production of spermatids, deformed spermatozoa and age-dependent decline in male fertility. We demonstrate that young long-interspersed nuclear elements (LINE1) and endogenous retroviral elements (ERVs) subfamilies, were upregulated in meiotic spermatocytes of Daxx null testes. Further study found that the Daxx mutant meiotic cells exhibit DNA hypomethylation in TEs. In summary, we have identified DAXX as a previously unknown key regulator of spermatogenesis that may function as an epigenetic regulator to silence young LINE1 by DNA methylation.
Project description:For proteomics analysis, testes of Ube2j2-/- and WT mice were collected on ice, washed once with pre-cooled PBS, dried with absorbent paper, and immediately placed in liquid nitrogen, dry ice, or at -80 ℃. Samples were packed in dry ice and transported to Hangzhou Jingjie Biotechnology Company (PTM Biolabs, Inc.) for whole protein 4D label-free quantitative proteomic determination. Testes of Ube2j2-/- and WT mice were subjected to LC-MS/MS analysis following a standard protocol. The sample was grinded and mixed with lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail III (Merck Millipore, 539134) dissolved in aqueous solution containing buffer salts), followed by sonication on ice using a high intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Then, the supernatant was collected and the protein concentration was determined with BCA kit (Beyotime Biotechnology, P0011) according to the manufacturer’s instructions. The sample was slowly added to a final concentration of 20% (m/v) trichloroacetic acid (Sigma-Aldrich, T4885) to precipitate proteins, then mixed by vortexing and incubated for 2 h at 4 °C. The precipitate was collected by centrifugation at 4500 x g for 5 min at 4°C. The precipitated protein was washed 3 times with pre-cooled acetone and dried for 1 min. Protein samples were then redissolved in 200 mM TEAB (Triethylammonium bicarbonate buffer, Sigma-Aldrich, T7408) and ultrasonically dispersed. Trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight. The sample was reduced with 5 mM dithiothreitol for 60 min at 37 °C and alkylated with 11 mM iodoacetamide for 45 min at room temperature in darkness. Finally, the peptides were desalted using a Strata X SPE column (Phenomenex). The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). The released peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Maxquant 1.6.15.0 software was used for spectra analysis, database searches, and peptide identification.
Project description:Cisplatin is a crucial chemotherapeutic agent for treating various cancers, but excessive use can cause irreversible damage to the reproductive system. However, the protein expression profile of cisplatin-induced testicular injury remains poorly understood. In this study, we observed that cisplatin treatment resulted in smaller testes, reduced sperm count, and a significant decrease in the number of spermatocytes and spermatids in mice. Further label-free quantitative proteomic analysis revealed that cisplatin reduced the expression of CDK1 in the testes, thereby affecting the number of spermatocytes and spermatids. These findings provide new insights into fertility preservation for cancer patients undergoing chemotherapy.
Project description:Pachytene spermatocytes were isolated from adult wild-type and Hsf5 knockout mouse testes using STA-PUT velocity sedimentation. Single-cell suspensions were prepared by enzymatic digestion of seminiferous tubules with collagenase and trypsin. Cells were loaded onto a 4%–2%–0.5% BSA gradient and allowed to sediment for 1 hour and 45 minutes at 4°C. Fractions were collected, and pachytene spermatocytes were identified by SYCP3, γH2AX, and DAPI staining under microscopy. Enriched pachytene cells were pooled and processed for RNA-Seq.
Project description:Active enhancers are identified by H3K27ac ChIP-seq analysis. To determine the dynamics of active enhancers during spermatogenesis, we performed H3K27ac ChIP-seq and detected reagions of active enhancers during spermatogenesis. We analyzed four representative stages of spermatogenesis: Thy1+ undifferentiated spermatogonia, which contains spermatogonial stem cells and progenitor cells; c-Kit+ differentiating spermatogonia from P7 testes; purified pachytene spermatocytes (PS) undergoing meiosis; and postmeiotic round spermatids (RS) from adult testes.
Project description:To determine the dynamics of open chromatin at a genomic resolution during spermatogenesis, we performed ATAC-seq and detected genomic regions of accessible chromatin by Tn5 transposase during spermatogenesis. We analyzed four representative stages of spermatogenesis: Thy1+ undifferentiated spermatogonia, which contains spermatogonial stem cells and progenitor cells; c-Kit+ differentiating spermatogonia from P7 testes; purified pachytene spermatocytes (PS) undergoing meiosis; and postmeiotic round spermatids (RS) from adult testes
Project description:Investigation of whole genome gene expression level changes of testes in the meiotic drive system in aedes aegypti during spermatogenesis compared to non drive strain. The meiotic drive system in Aedes aegypti causes the female determining chromosome to fragment during spermatogenesis. A six chip study using total RNA from three separately extracted non driving strain testes of Aedes aegypti and three separately extracted meiotic drive strain testes of Aedes aegypti.