Dataset Information

Proteomics of isolated rat manchettes

ABSTRACT: The manchette is a transient microtubular structure that plays a vital role in nuclear shaping during spermiogenesis. It comprises thousands of microtubules (MTs) that build a scaffold surrounding the distal half of the nucleus. The manchette distributes proteins and vesicles during spermiogenesis in a process called intra-manchette transport (IMT). The current hypothesis is that IMT shares many similarities with intra-flagellar transport (IFT) and utilizes both MTs and filamentous actin (F-actin). However, IMT is still poorly understood as direct visualization of IMT complexes is missing, and the presence of F-actin has not been experimentally shown. Here, we use cryogenic-electron tomography (cryo-ET) to visualize IMT components in manchettes extracted from rat spermatids. Additionaly, we used proteomics to identify and quantify IMT composition. We find that F-actin is an integral part of the manchette with two different spatial organizations, namely bundles and single filaments, indicating two different functional roles. We further uncover that IMT on MTs is mediated by two distinct transport machineries: dynein mediated transport of soluble cargo and dynein independent transport for vesicles. Our results provide new insights into the manchette's function as a transport scaffold, highlighting its significance for the polarization of spermatids during spermiogenesis.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Testis, Sperm

SUBMITTER: Laura Pérez Pañeda

LAB HEAD: Albert J.

PROVIDER: PXD055902 | Pride | 2025-07-18

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20231106_uniprotkb_proteome_UP000002494_AND_revi_withisoforms.fasta	Fasta
	20231113_uniprotkb_proteome_UP000002494_AND_isoforms_AND_unreviewed.fasta	Fasta
	20240126_EX3_UM5_perez044_SA_EXT00_Manchette_clean1.raw	Raw
	20240126_EX3_UM5_perez044_SA_EXT00_Manchette_clean2.raw	Raw
	20240126_EX3_UM5_perez044_SA_EXT00_Manchette_clean3.raw	Raw

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Project description:Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis and metastasis of tumor cells. The testis specific PFN3 is localized in the acroplaxome-manchette complex of developing sperm. We demonstrate, that PFN3 further localizes during spermiogenesis in the Golgi complex and proacrosomal vesicles, suggesting a role in acrosome formation. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. Using CRISPR/Cas9 genome editing we generated mice deficient for Pfn3. Pfn3-/- males are sub-fertile displaying a type II-globozoospermia. We revealed, that Pfn3-/- sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3-/- sperm showed reduced sperm motility resulting from flagellum deformities. We show, that acrosome biogenesis is impaired starting from the Golgi phase. RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence mTOR was activated and AMPK was suppressed resulting in the inhibition of autophagy. This dysregulation of AMPK/ mTOR affected the autophagic flux which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. We conclude that this disruption leads to the observed malformation of the acrosome. Further, actin related protein ARPM1 was absent in the nuclear fraction of Pfn3-/- testis and sperm. This suggests, that lack of PFN3 leads to destabilization of the stable PFN3-ARPM1 complex resulting in the degradation of ARPM1. Interestingly, in the Pfn3-/- testis, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2) and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicates this protein as a candidate for male factor infertility in humans.

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Proteomics of isolated rat manchettes

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