Molecular Background of Male Infertility

In a recent mass spectrometry analysis, we were able to determine proteins of about 7,000 genes in human sperm. This is quite a lot considering that expression of less than 20,000 protein-coding genes in the human genome has been verified at the protein level so far. In addition, we had compared protein abundances between sperm of normal-fertile men and infertile men diagnosed with reduced sperm count or oligozoospermia (monosymptomatic and syndromatic). We found abundances of many proteins (actually protein groups) to be reduced in association with oligozoospermia relative to normozoospermia. According to gene ontology analysis, these proteins are important for proper spermatogenesis and sperm functioning. As shown in the below pie chart, prevalent functional involvements were refering to the cilium or sperm flagellum including the axoneme. Thus, reduced sperm count might not be the only reason for fertility impairment in cases of oligozoospermia. An additional factor could be that the fewer sperm are dysfunctional to a higher proportion (Greither et al. 2023).

The study was conducted together with colleagues from the Institute of Molecular Biology Mainz and the Center for Reproductive Medicine and Andrology at the University Hospital Halle (Saale), and was a Top Viewed Article in the 12 months after publication (Dec 2023). Furthermore, it was awarded as the Best Original Research Article by the Journal Andrology on the occasion of the Joint Congress of the American Society of Andrology & International Congress of Andrology 2025.

Functional Relevance of Proteins for Male Fertility Maintenance

We are investigating the molecular causes of male infertility which affects millions of couples worldwide. Part of our research addesses which genes might have increased importance for the maintenance of male fertility? As an approximation, we have assessed the Fertility Relevance Probability of thousands of proteins and their coding genes. For each of these genes, we have calculated a score which integrates clinical manifestations, phenotypes of knockout mice, levels of gene sequence conservation, transcript abundance, and protein interconnectivity. The score can take any value from zero to 1.0 whereby higher values suggest higher relevance for male fertility maintenance. For example, the top-ranked score for the gene encoding AKAP4 in the below table section suggests a particularly high importance for proper sperm functioning. The detailed results are presented on the website PreFer Genes (Greither et al. 2020).

The Impact of Mating Systems on Sperm Protein Evolution

We have examined the evolution of primate sperm proteins in the light of species-specific levels of competition between males. Our analyses revealed rather low evolutionary rates of the genes coding for sperm zonadhesin (ZAN) and sperm adhesion protein 1 (SPAM1) in species exhibiting strong sexual dimorphism of body weight. The below figure illustrates this in the example of SPAM1, which is an enzyme aiding the sperm to penetrate the cumulus cell layer surrounding the oocyte. According to our findings, the rate ratio of amino acid-altering to silent substitutions in the coding gene (dN/dS) is overall lowered in species with higher sexual size dimorphism (male weight/female weight). This is particularly evident in primate species in which harem formation occurs (red datapoints). Here, the dominant male more or less successfully monopolizes female matings, also by physical force. Thus, there will be a comparably smaller level of competition between sperm of different males within the female genital tract. Selective pressure for raising male competitiveness through improved functioning of sperm proteins such as SPAM1 should be low. In the other species (blue datapoints), harem formation should not play a major role. Here, higher dN/dS values might reflect selection pressure on SPAM1 for improved properties and/or relaxation of so-called functional constraint. We observed basically the same pattern in primate genes coding for ZAN (Herlyn and Zischler 2007; Prothmann et al. 2012).

Effects of Amino Acid Exchanges on Protein Properties

We examined amino acid exchanges that occur in interdependence within single proteins. In the corresponding study, we had included a measure of intra-protein co-evolution, node degree and hydropathy. The latter two parameters were intended to approximate the extend of outward-directed interactions with other proteins (node degree) and of engaging in inward-directed structure formation (hydropathy). The fourth parameter, dN, should give the non-synonymous substitution rate. In the below figure, blue connections highlight a persistent positive correlation between our measure of within-protein co-evolution and the non-synonymous substitution rate (dN), which is reasonable since the first is part of the latter. Our findings further suggest that within-protein co-evolution is all the more directed outward, the closer a protein acts to fertilization. This is evidenced in the below figure by a growing number of negative correlations (red edges) from liver-expressed genes and their proteins (LIVER1, LIVER2) via samples representing entire body (BODY1, BODY2) to genes coding for testis (TESTIS) and sperm proteins (SPERM). In fact, if sperm proteins with particularly many amino acid sites that evolve in interdependence have less interaction partners (lowered node degree) and engage less in their own structure formation (decreased hydropathy), their functional focus should be primarily directed outward (Kwiatkowski et al. 2020). For example, the evolution of a sperm ligand like above-mentioned ZAN should primarily reflect its outward interaction with a female receptor protein.

Toward a More Comprehensive Picture of Evolutionary Forces Acting on Sperm and Testis Proteins

Phosphorylation is an important player in the control of protein function. This let us examine phosphorylation patterns in human sperm proteins using two-dimensional gel electrophoresis and Western blotting. We observed overall lowered evolutionary rates in genes coding for sperm phosphoproteins compared to their counterparts encoding non-phosphorylated sperm proteins. Apparently, selection counteracts amino acid exchanges in sperm phosphoproteins. This makes sense since exchanges would usually interfer with established protein activity patterns and the more numerous interactions of sperm phosphoproteins with other proteins (Schumacher et al. 2013). A follow-up investigation substantiated a general trend of increasing numbers of protein interactants with stronger sequence conservation in primate genes coding for sperm proteins. The same study emphasized the impact of mating system variation on evolutionary rates (e.g., Schumacher et al. 2014). In yet another study, we have shown that genes coding for testicular and sperm proteins are all the more conserved the higher their evolutionary age. In fact, higher gene age usually associates with basic cellular functions which mostly have to be maintained (Schumacher et al. 2017; Schumacher & Herlyn 2018). A better knowledge of the diverse factors affecting protein evolution might prove useful for more precise predictions of the functional relevance of testicular and sperm proteins for the maintenance of male fertility.