Dataset Information

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm

ABSTRACT: Human sperm cell were analysed for their complete protein content and a few proteins of interest were absolutely quantified with labelled standard peptides and targeted mass spectrometry.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Spermatozoon, Sperm

SUBMITTER: Svenja Victoria Kaufmann

LAB HEAD: Henning Urlaub

PROVIDER: PXD036819 | Pride | 2023-09-12

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	M_Ninov_BKaupp_Sperm_070817_140220_Expl_bRP_1.raw	Raw
	M_Ninov_BKaupp_Sperm_070817_140220_Expl_bRP_10.raw	Raw
	M_Ninov_BKaupp_Sperm_070817_140220_Expl_bRP_11.raw	Raw
	M_Ninov_BKaupp_Sperm_070817_140220_Expl_bRP_12.raw	Raw
	M_Ninov_BKaupp_Sperm_070817_140220_Expl_bRP_13.raw	Raw

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Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm.

Grahn Elena E Kaufmann Svenja V SV Askarova Malika M Ninov Momchil M Welp Luisa M LM Berger Thomas K TK Urlaub Henning H Kaupp U Benjamin UB

Nature communications 20230905 1

The reaction of CO2 with H2O to form bicarbonate (HCO3-) and H+ controls sperm motility and fertilization via HCO3--stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3- in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The rest ...[more]

PMID: 37669933

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Project description:It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilisation. However, the function of the sperm transcriptome remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are not likely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilisation and can be detected at least until the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular a highly significant enrichment for transcripts encoding Ribosomal Proteins. The identification of a conserved set of spermatozoal transcripts opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules. RNA extracted from three biological replicates of purified sperm (Sperm rep1, Sperm rep2 and Sperm rep3) was used as a template for oligo-dT-primed reverse transcription, amplification, labelling of dye swapped technical replicates and hybridisation to long oligonucleotides microarrays. As a control, RNA from two biological replicates of dissected adult testis plus accessory glands (Testis_rep1, Testis_rep2) was amplified, labelled (dye-swap technical replicate) and hybridised to similar arrays. To help with the spot-finding of the arrays genomic DNA was co-hybridised in some cases (this genomic DNA data was excluded from further analysis). Genes with an intensity level below 200 in at least one channel across the Sperm or Testis set were removed (5579 transcripts present in all three sperm replicates, 5358 transcripts from the testis/accessory gland samples and 4295 transcripts common to both data sets). Then the quantile normalisation was independently applied to the Sperm replicate samples and Testis replicates.

Project description:BACKGROUND: In previous studies using candidate gene approaches, low sperm count (oligospermia) has been associated with altered sperm mRNA content and DNA methylation in both imprinted and non-imprinted genes. We performed a genome-wide analysis of sperm DNA methylation and mRNA content to test for associations with sperm function. METHODS AND RESULTS: Sperm DNA and mRNA were isolated from 21 men with a range of semen parameters presenting to a tertiary male reproductive health clinic. DNA methylation was measured with the Illumina Infinium array at 27,000 CpG loci. Unsupervised clustering of methylation data differentiated the 21 sperm samples by their motility values. Recursively partitioned mixture modeling (RPMM) of methylation data resulted in four distinct methylation profiles that were significantly associated with sperm motility (P=0.01). Linear models of microarray analysis (LIMMA) was performed based on motility and identified 9,189 CpG loci with significantly altered methylation (Q<0.05) in the low motility samples, with many loci located in genes associated with subfertility and epigenetic regulation. In the low motility samples, the majority of disrupted CpG loci (80%) were hypomethylated. Of the aberrantly methylated CpGs, 194 were associated with imprinted genes almost equally distributed into hypermethylated (predominantly paternally expressed) and hypomethylated (predominantly maternally expressed) groups. Sperm mRNA was measured with the Human Gene 1.0 ST Affymetrix GeneChip Array. LIMMA analysis based on motility identified 20 candidate transcripts as differentially expressed in low motility sperm, including HDAC1 (NCBI 3065), SIRT3 (NCBI 23410), and DNMT3A (NCBI 1788). Altered expression of these epigenetic regulatory genes was associated with RPMM DNA methylation class. CONCLUSIONS: Using integrative genome-wide approaches to study epigenetic and gene expression patterns in human sperm we identified CpG methylation profiles and mRNA alterations associated with low sperm motility, and that low motility sperm may have aberrant genome-wide hypomethylation due to excess HDAC1 activity. See summary above

Dataset Information

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm

Dataset's files

Publications

Control of intracellular pH and bicarbonate by CO<sub>2</sub> diffusion into human sperm.

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