Spermiogenesis alterations in the absence of CTCF revealed by single cell RNA sequencing

Abstract

CTCF is an architectonic protein that organizes the genome inside the nucleus in almost all eukaryotic cells. There is evidence that CTCF plays a critical role during spermatogenesis as its depletion produces abnormal sperm and infertility. However, defects produced by its depletion throughout spermatogenesis have not been fully characterized. In this work, we performed single cell RNA sequencing in spermatogenic cells with and without CTCF. We uncovered defects in transcriptional programs that explain the severity of the damage in the produced sperm. In the early stages of spermatogenesis, transcriptional alterations are mild. As germ cells go through the specialization stage or spermiogenesis, transcriptional profiles become more altered. We found morphology defects in spermatids that support the alterations in their transcriptional profiles. Altogether, our study sheds light on the contribution of CTCF to the phenotype of male gametes and provides a fundamental description of its role at different stages of spermiogenesis.

Keywords: CTCF; ScRNA-seq; mouse testis; sperm; spermatogenesis.

FIGURE 1

Clustering, validation, and identification of spermatogenesis populations from WT and Ctcf-cKO testes. (A) UMAP visualization of 10 cell clusters from WT and Ctcf-cKO cell populations. (B) Visualization of cell clusters by genotype. Clusters are distinguished by color according to the key color. (C) Heatmap displaying the percentage of similarity shared between our 10 clusters versus 17 clusters obtained in Hermann, et al., 2018 study. We considered a p-value adjusted <0.05 in genes list from each cluster. Cluster identity is mentioned to the right of the heatmap. (D) Dot plot of the proportion of cells in the respective cluster expressing each marker (dot size), and the average expression (color scale).

FIGURE 2

Inferring cell trajectory and functional annotation. (A) Pseudotime analysis in spermatogenic cells. Scale color in pseudotime shows the trajectory in specialization cell process. The projection was performed using UMAP and the parameter k = 3 in monocle3 1.2.6. (B) UMAP projection without somatic cells and molecular markers identification of cells populations. (C) Line time of spermatogenesis process with location of clusters obtained in this dataset based on similarity heatmap, molecular markers identification and pseudotime analysis. E-RS = Early-Round Spermatids, M-RS = Mid-Round Spermatids, L-RS = Late-Round Spermatids. (D) Differential gene expression (p-value adjusted <0.05) performed between Ctcf-cKO against WT cells in each cluster, and the top 5 GO terms of the upregulated (green boxes) and downregulated genes (yellow boxes) in the different cell clusters.

FIGURE 3

Gene Set Enrichment Analysis (GSEA) of Ctcf-cKO against WT spermatogenesis cells and differential gene expression of key players in histones replacement. (A–E) Positively enriched maps of altered biological functions in Ctcf-cKO versus WT cells. The comparisons were made in each cluster. Each green circle (node) represents a pathway. A link between pathways was established when 2 pathways had a Jaccard index >0.4, given the expressed genes. All enriched pathways have an FDR <0.05 and a p-value <0.01. (F) Violin plots of the expression profiles of Tnp1, Tnp2, Prm1, and Prm2 in round spermatids of Ctcf-cKO and WT within cell clusters 3, 2 and 0. Statistical differences are based on the Wilcoxon rank sum test, asterisks indicated p < 0.05, expression levels correspond to log normalized values.

FIGURE 4

Immunofluorescent staining pattern of TNP1, TNP2, and PRM2 in WT and Ctcf-cKO testis sections. (A–E) Show simultaneous immunodetection of TNP1 and TNP2 in elongated spermatids from WT testis sections. (F–J) Simultaneous immunodetection of TNP1 and TNP2 in elongated spermatids from Ctcf-cKO testis sections. Elongated spermatids with immunodetection of both proteins are shown with arrowheads, while abnormal spermatids with signal only for TNP1 are shown with asterisks. 25% of the analyzed seminiferous tubules showed spermatids with an abnormal staining pattern. Three mice of each genotype were analyzed.

FIGURE 5

Immunofluorescent staining pattern of PRM1 and PRM2 in WT and Ctcf-cKO testes sections. (A–D) PRM1 immunodetection in elongated spermatids from WT testes sections. Some representative elongated spermatids with PRM1 signal are pointed out with arrowheads. (E–H) Immunodetection of PRM1 in elongated spermatids from Ctcf-cKO testis sections. Elongated spermatids with and without the PRM1 signal are pointed out with arrowheads and arrows, respectively. (I–L) Immunodetection of PRM2 in elongated spermatids from WT testis sections. (M–P) Immunodetection of PRM2 in elongated spermatids from Ctcf-cKO testis sections. Elongated spermatids with PRM2 immunodetection are shown with arrowheads.

FIGURE 6

Electron micrographs of spermatids from WT and Ctcf-cKO mice. (A) Micrograph showing the acrosome sac (red line) and head cap (black bar) of a WT round spermatid. (B) Abnormal acrosome sac (red dashed line) and head cap (black bar) of a round Ctcf-cKO spermatid. (C) Head cap (black circle) from a WT elongating spermatid. (D) Abnormal head cap (black dashed circle) of an elongating Ctcf-cKO spermatid. (E) Acrosome in the anterior apical end of elongated spermatids from WT mice (red double head arrow). (F) Misplaced acrosome (red dashed double head arrow) in an elongated spermatid from Ctcf-cKO mice. (G) Acrosome at the anterior apical end of the elongated spermatids of the WT testes (red arrows). (H) Abnormal acrosome and altered morphology in elongated spermatids from testis in Ctcf-cKO mice (red arrows). (I, J) Proper nuclear morphology and chromatin compaction in round spermatids from WT and Ctcf-cKO mice, respectively. (K) Elongating spermatid with electron-dense material associated with the nuclear envelope (blue arrows) from WT mice. (L) Elongating spermatid with discontinuities in the dense structure associated with the nuclear envelope and in the nuclear envelope itself (blue arrows) from Ctcf-cKO mice. (M) Elongated spermatid with condensed chromatin (blue bar) from WT mice. (N) Elongated spermatid with abnormal chromatin condensation pattern of Ctcf-cKO mice (blue dashed line). (O) Elongated spermatid displaying full chromatin packing inside the nucleus (blue contoured line) from WT mice. (P) Elongated spermatid displaying apparently full chromatin packing (blue contoured dashed line), but with abnormal head morphology from Ctcf-cKO mice. Scale bars represent 2 μm.