Dataset Information

Dynamic sperm proteome remodelling during epididymal maturation in a marsupial, Macropus giganteus

ABSTRACT: While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile changes over epididymal maturation. Samples prepared by FASP digestion were analysed by LC-MS/MS with SWATH acquisition. A total of 4,304 proteins were identified, with significant overlap across epididymal regions. Highly abundant proteins in common across caput, corpus and cauda spermatozoa had strong enrichment for tubulins and included 4 histone proteins. The most significant proteomic remodelling was observed in the corpus to cauda transition, late in epididymal transit (728 differentially abundant proteins). Overall proteomic changes across epididymal maturation (1,131 differentially abundant proteins) suggested a loss of sperm glycosidases and an increase in flagellar proteins, including tubulins and dyneins. These findings serve to highlight both consistencies with eutherian sperm epididymal maturation (e.g. bias towards protein loss over transit, transfer of proteins via extracellular vesicles) and elements which are likely unique to marsupials (e.g. reduced chromatin stability, potential use of β-oxidation as a major metabolic pathway). This critical information can now be leveraged to further develop ARTs in marsupials.

INSTRUMENT(S):

ORGANISM(S): Macropus Giganteus

TISSUE(S): Spermatozoon, Sperm

SUBMITTER: Taylor Pini

LAB HEAD: Taylor Pini

PROVIDER: PXD064577 | Pride | 2025-09-29

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2300707_DIA_R1_caput.wiff	Wiff
	2300707_DIA_R1_caput.wiff.scan	Wiff
	2300707_DIA_R1_cauda.wiff	Wiff
	2300707_DIA_R1_cauda.wiff.scan	Wiff
	2300707_DIA_R1_corpus.wiff	Wiff

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Publications

Dynamic sperm proteome remodeling during epididymal maturation in a marsupial, Macropus giganteus†.

Kunkel Sarah S Johnston Stephen D SD Nouwens Amanda A Pini Taylor T

Biology of reproduction 20250901 3

While assisted reproductive technologies (ARTs) are widely used in domestic animals, successful implementation of ARTs to conserve wildlife species remains challenging. In macropods, crucial aspects of fundamental reproductive biology, including changes induced by epididymal maturation, remain unknown, limiting the development of ARTs. In this context, we performed a proteomic analysis of spermatozoa from the caput, corpus, and cauda epididymis of Eastern Grey Kangaroos (n = 6) to profile change ...[more]

PMID: 40689558

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Project description:Mammalian spermatozoa acquire their fertilizing ability during epididymal transit. Gene expression patterns along the epididymis are established by specific transcription factor networks that coordinate region-specific functions. The epididymis is usually divided into 3 segments: caput, corpus, and cauda. The human epididymis anatomy does not allow clear distinction between these three segments. To determine to which extent gene expression is segmented along the human epididymis, transcriptome profiling was performed on 8 distinct epididymal regions from 3 donors. Microarray analysis was performed on a Gene Chip Human Clariom S (Affymetrix®) array representing 337 100 transcriptional variants encoded by 20 800 genes. Proximal segments 1 to 3 were distinguishable from the distal epididymal segments (4 to 8) as shown by unsupervised Principal Component Analysis. Transcripts from each segment with differentially expressed genes (DEGs) > 2-fold change and FDR < 0.05 were clustered in relation to their intensity profiles. While no DEGs were detected between segments 1–3 corresponding to the efferent ducts, 1140 DEGs were detected between efferent ducts (1–3) and the epididymis (4–8), 400 between caput (4–6) vs. corpus/cauda (7–8) and none between corpus (7) and cauda (8). Gene Ontology annotation revealed that up-regulated DEGs in the efferent ducts (1–3) were predominantly related to cilium assembly/movement and cell differentiation. The biological process terms fertilization, defense and immune responses were associated with caput epididymis (4–6) while spermatogenesis and protein binding were found all along the epididymis (4–8). In conclusion, the proximal human epididymis is exclusively occupied by efferent ducts with a distinct DEG profile compared with the downstream epididymal segments. Moreover, gene expression profiling revealed two regions in the human epididymis; the caput and the distal corpus/cauda region. Taken together, analysis of the human epididymal transcriptome reveals a limited DEG profile.

Project description:Purpose: To compare the transcriptome profiles (RNA-seq) of cultured human epididymis cells and tissue from the caput, corpus and cauda regions of the human epididymis. Methods: Human epididymis tissue was obtained with Institutional Review Board approval from 3 patients (UC05, UC06, UC09, range: 22 - 36 years) undergoing inguinal radical orchiectomy for a clinical diagnosis of testicular cancer. None of the epididymides had extension of the testicular cancer. The three anatomical regions: caput, corpus and cauda, were separated and segments of each snap frozen. Adult human epididymis epithelial (HEE) cultures were also established from tissue. RNA was extracted from both tissue and cultured HEE cells and RNA-seq libraries prepared (TruSeq RNA Sample Preparation Kit v2, Low-Throughput protocol, Illumina). Libraries were sequenced on Illumina HiSeq2500 machines. Data were analyzed using TopHat and Cufflinks. Results: Libraries generated ~19-39 million reads per library from the cells (95-99% mapping to the human genome) and ~14-39 million reads from the tissue samples (84-99% mapped). Raw reads were aligned to the genome with Tophat and gene expression values were processed using Cufflinks as Fragments Per Kilobase per Million mapped fragments (FPKM). FPKM values were subject to principle component analysis, which revealed that though caput, corpus and cauda cell samples respectively from UC05, UC06 and UC09 clustered together. RNA-seq data from the 3 biological replicas (UC05, UC06 and UC09) of caput, corpus and cauda were pooled for further analysis. Cufflinks was used to determine differentially expressed genes (DEGs) between caput, corpus and cauda cells, combined from the 3 donors. The gene expression profiles of corpus and cauda are remarkably similar and both differ from the caput to a similar degree. We identified ~40 genes differentially expressed between corpus and cauda and more than 1600 DEGs between caput and cauda. The DEGs for each comparison (caput and corpus/cauda) were analysed using a gene ontology process enrichment analysis (DAVID, Huang et al., NAR 2009;37:1-13, Huang et al., 2009 Nat Prot 4:44-57). Conclusions: Here we describe an in depth analysis of the gene expression repertoire of primary cultures of epithelial cells and intact tissues from each region of the adult human epididymis. These data will be valuable to decipher pathways of normal epididymis function and aspects of epididymis disease that cause male infertility. RNA-seq was performed on libraries generated from caput, corpus and cauda-derived cultured cells (passage 2 or 3) from 3 donors and on caput, corpus and cauda tissue from 2 of the same donors. Donor age range: 22 - 36 years.