A Quantitative Mass Spectrometric Analysis of Signalling Proteins in Human Sperm Cells
by Svenja Victoria Kaufmann
Date of Examination:2024-03-27
Date of issue:2024-05-17
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Ivo Feussner
Persistent Address:
http://dx.doi.org/10.53846/goediss-10484
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Abstract
English
Fertilization of an oocyte by a sperm cell is a complex multi-stage process. First, a sperm cell must locate and reach the oocyte; second, it must undergo capacitation, the acrosome reaction, and hyperactivation to penetrate the oocyte’s investment; finally, the membranes of sperm and oocyte fuse with each other. Many hypotheses about fertilization in humans have been proposed based on studies conducted on non-human organisms. Recently, it became clear that concepts developed in one species do not hold for other species. Proteins involved in the fertilization process are often species-specific and even orthologues from closely related species differ in properties and functions. Furthermore, signalling concepts developed for somatic cells have guided fertilization research. However, many sperm proteins are not only species-specific, but often also sperm-specific, i.e., they do not exist in somatic cells and vice versa. Inevitably, this has led to many controversies. I used different mass spectrometry (MS) techniques to investigate the protein inventory and signalling mechanisms of human sperm. Using targeted MS, I have determined the copy number of proteins involved in bicarbonate, pH, Ca2+, and cAMP signalling. These experiments revealed an extraordinarily high dynamic range of protein expression in human sperm. Some proteins are expressed with a few thousand molecules per cell such as the sperm-specific Ca2+ channel CatSper, whereas others are expressed with several million copies such as the A-kinase anchoring protein AKAP4. The carbonic anhydrase 2 (CA2) is expressed with ~150,000 molecules/cell and was identified as a key molecule in bicarbonate and pHi homeostasis. Additionally, I could not detect many proteins that were previously identified as key players in fertilization. For these proteins, I established the limit-of-detection (LOD) approach, which allows to determine the maximal copy number of a protein. Using this LOD approach, I estimate that proteins that were not detected in human sperm have maximal copy numbers of about < 10 copies/cell; I will discuss why such low copy numbers are difficult to reconcile with a physiological function. Specifically, the solute carriers SLC4A4 and SLC26A6, the cystic fibrosis conductance regulator (CFTR), and the amiloride-sensitive epithelial sodium channel (ENaC), seemingly involved in HCO3- transport, featured such low copy numbers. The LOD approach was also used to estimate the copy number of putative thermosensors involved in sperm thermotaxis. Several members of the transient receptor potential channels as well as various rhodopsin forms also displayed very low LOD values. Finally, signalling mechanisms involving cAMP were investigated with phospho-peptide enrichment techniques and MS. An inventory of phosphorylated sites in human sperm was assembled, and phospho-sites in proteins that are regulated during capacitation were identified. For this, the phosphorylation pattern of human sperm cells stimulated with either bicarbonate or human serum albumin (HSA) was compared with unstimulated cells. Stimulation with bicarbonate led to tyrosine phosphorylation, which has recently been refuted as a pre-requisite for capacitation. And, both stimulation with bicarbonate and with HSA led to serine phosphorylation of the A-kinase anchoring proteins AKAP3 and AKAP4, fibrous sheath interacting protein 2 (FSIP2), outer dense fiber proteins (ODF), and the mitochondria-eating protein (SPATA18). The network of AKAP3, AKAP4, FSIP2, and ODF1/2 has previously been associated with regulatory phosphorylation processes in human sperm. The results of the phosphorylation analysis strongly suggest that AKAP3 and AKAP4 are at the core of regulatory processes.
Keywords: Human sperm; Quantitative Mass Spectrometry; Proteome; Phosphoproteome; Capacitation
