Dataset Information

Intercellular bridges are essential for transposon repression and meiosis in the male germline

ABSTRACT: Germ cell connectivity via intercellular bridges is a widely conserved feature but its functional significance is poorly understood. Intercellular bridges are essential for fertility in male mice as genetic ablation of a critical bridge component, TEX14, causes loss of germ cell connectivity and meiotic failure, however the underlying reasons are unknown. A screen for meiosis-defective mouse mutants yielded a sterile mutant harboring a point mutation in Tex14 that leads to a partial loss of germ cell intercellular bridges. We utilized the Tex14 hypomorphs with reduced intercellular bridges, along with Tex14-null mice that completely lack bridges to examine the roles of germ cell connectivity during adult spermatogenesis. Complete or partial loss of intercellular bridges does not severely deplete spermatogonia, but spermatocytes are unable to undergo meiotic DNA replication and enter meiotic prophase. Phenotypic severity correlates with the extent of intercellular bridge depletion, with Tex14 hypomorphs containing a relatively larger number of spermatocytes undergoing meiosis, thus allowing us to assess impacts on meiotic progression. Tex14-deficient germ cells that enter prophase form meiotic breaks and initiate synapsis, but later exhibit synaptic failures and retain unrepaired breaks. Strikingly, Tex14-deficient germ cells derepress LINE1 retrotransposons and accumulate LINE1-encoded proteins during meiosis. Single-cell RNA-sequencing of testes from Tex14 hypomorphs show that intercellular bridge depletion modestly impacts the RNA landscape in early germ cell stages, while mutant cells that progress to late spermatocyte and post-meiotic spermatid stages broadly upregulate retrotransposons and dysregulate gene expression. RNA-sequencing confirms extensive sharing of transcripts between normal spermatids and demonstrates its partial attenuation in Tex14 hypomorphs, indicating that intercellular bridges enable exchange of cytoplasmic material between connected germ cells in testes. Tex14 hypomorphs display a progressive decline in germ cell numbers starting from the spermatocyte stage, with a complete lack of elongating spermatids, and are therefore sterile. Our findings reveal that the regulation of meiosis is non-cell-intrinsic and inform a model in which intercellular bridges orchestrate critical meiotic events and protect germline genome integrity during spermatogenesis, possibly by facilitating the exchange of regulatory factors.

ORGANISM(S): Mus musculus

PROVIDER: GSE274959 | GEO | 2025/01/06

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Purpose: Investigate the transcriptomes of Tex14 heterogygous sample (with intercellular bridges intact) compared to Tex14 homozygous mutant (lacking intracellular bridges) to ascertain what effects the loss of cytoplasmic sharing has on mitotic to meiotic mouse ovary Methods: Single cell RNA sequencing libaries were created from embryonic day 13.5 mouse ovaries from Tex14 +/- and Tex14 -/- samples. Three Tex14+/- and two Tex14-/- E13.5 ovaries were pooled. Libaries were creating using 10X Chromium Single Cell 3' (v2 Chemistry) library kits per 10X posted protocols. Samples were sequenced on HiSeq 4000 (Illumina) with paired-end sequencing parameters: Read1, 98 cycles; Index1, 14 cycles; Index2, 8 cycles; and Read2, 10 cycles. Tex14+/- sample had 23,567 mean reads per cell, and Tex14-/- had 19,735 mean reads per cell. Since 10X Genomics recommends an average of 60k raw reads per cell, we submitted our two libaries for deeper sequencing. Our second sequencing run was done on a Novaseq S2 flowcell with the following parameters: Read 1 Length 26bp; Index 1 Length 8bp; Index 2 Length 0bp; Read 2 Length: 98 bp. Follwing deeper sequencing, Tex14+/- sample had 55,744 mean reads per cell, and Tex14-/- had 41,117 mean reads per cell. Cells which passed quality control metrics were further analyzed. Results: We used CellRanger v2.1.0 to align the sequenced data to the 10X Genomics pre-built mm10 genome. We mapped about 300,000,000 sequence reads for each shallowly sequenced library, and 750-800,000,000 reads for the deeply sequenced libraries. We used Seurat v3 to keep high quality cells, excluding cells which did not all of the following metrics: genes expressed in at least 3 cells, fewer than 25,000 unique fragments (to exclude cell doublets), greater than 200 genes expressed (to exclude GEMS with no cells), and cells with 7.0% or less mitochondrial gene expression. After quality control exclusion, cell counts for the gene by cell matrices are as follows: tex14 het shallow, 12260 cells; tex14 ko shallow, 14715 cells; tex14 het deep, 11794 cells; tex14 ko deep, 13982 cells. Conclusions: Our study represents the first single cell RNA sequencing experiment conducted on fetal mouse ovaries lacking intracellular bridges. We observed that the absence of Tex14 did not alter the transcriptional composition of ovarian somatic cells, but specifically attenuated the level of pluripotency-associated transcripts as germ cells enter meiosis.

Project description:The role of different proteins, Always Early (Aly), Spermatocyte Arrest (Sa), Ubi-p63E (Magn) on the gene expression in spermatocyte differentation was assessed by microarray ABSTRACT: The ubiquitin proteasome system (UPS) regulates many biological pathways by posttranslationally ubiquitinating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitinated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show that loss of function of Drosophila magellan (magn), the polyubiquitin Ubi-p63E, results in specifically meiotic arrest sterility in males. Expression of ubiquitin from magn/Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. Function of magn/Ubip63E is required cell autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn/Ubi-p63E mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I with lack of protein expression of key meiotic cell cycle regulators Boule and Cyclin B. Loss of function of magn/Ubi-p63E did not strongly affect the spermatocyte transcription program regulated by the tTAF and tMAC genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn/Ubi-p63E phenotype may not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest. RNA was obtained from whole testes of flies with following mutations: 1) aly[2]/aly[5p], 2) sa[1]/sa[2], 3) magn[12c]/magn[23b] or magn[12c]/magn[12c];pSC2, 4) red[1], e[1] (WT). Each genotype had three biological replicates except magn which had four total biological replicates, two from magn[12c]/[23b] and two from [12c]/[12c]; pSC2

Dataset Information

Intercellular bridges are essential for transposon repression and meiosis in the male germline

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets