Molecular Background of Male Infertility

In a recent mass spectrometry analysis, we were able to determine proteins of about 7,000 genes in the 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 that study, we compared protein abundances between sperm of normal-fertile men and infertile men diagnosed with reduced sperm count or oligozoospermia. This revealed that abundances of many proteins (actually protein groups) are reduced in oligozoospermia. According to the gene ontologies, these proteins are improtant for proper spermiogenesis and sperm functioning. In the below pie chart, this is evidenced by prevalent functional involvements in axoneme assembly and functioning etc. Thus, reduced sperm count might not be the only reason for fertility impairment in men with oligozoospermia. An additional factor could be that the fewer sperm are dysfunctional to a higher proportion (Greither et al. 2023). Notwithstanding this, assisted reproduction usually enables pregnancies in the couples affected. Furthermore, men with oligozoospermia can be fertile, whereas part of the men presenting with normozoospermia is not. This illustrates the many facets of male fertility impairment.

The study was conducted in close cooperation with colleagues at the Institute of Molecular Biology Mainz and the Center for Reproductive Medicine and Andrology at the University Hospital Halle (Saale), and 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. A central question is which genes have increased importance for the maintenance of male fertility? To answer this, we have assessed the Fertility Relevance Probability of thousands of proteins and their coding genes. For each gene, 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. The detailed results are presented on the website PreFer Genes (Greither et al. 2020). For example, the top-ranked gene coding for AKAP4 in the below screenshot is essential for proper sperm functioning.

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 get through 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 little 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 on SPAM1 for improved properties and/or relaxation of so-called functional constraint. Phylogenetic bias does not account for the pattern as we show in the corresponding paper. Rather, our results emphasize the impact of mating system variation on the evolution of sperm proteins. We observed basically the same in 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. Our findings suggest that the effect of such within-protein co-evolution is all the less inwardly directed, 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 particular, there are more an more negative correlations between within-protein co-evolution and hydropathy and node degree toward sperm proteins. Yet, if sperm proteins with particularly many co-evolving amino acid sites engage less in protein structure formation (decreased hydropathy) and have less interaction partners (lowered node degree), then probably because their functional focus is primarily directed outward. In fact, the evolution of a sperm ligand, for example, should primarily reflect its outward interaction with a female receptor protein. By the way, blue connections in the below figure 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 (Kwiatkowski et al. 2020).

Toward a Complete Picture of Evolutionary Conservation of Sperm and Testis Proteins

Phosphorylation is an important player in the control of protein function. This led us examine phosphorylation patterns in human sperm proteins using two-dimensional gel electrophoresis and Western blotting. Notably, 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 mostly interfer with established protein activity patterns and the 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 first of all have to be maintained (Schumacher et al. 2017; Schumacher and 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.