Klaus Steger

Protamine 2 (Prm2/PRM2), together with Protamine 1 (Prm1/PRM1), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange, a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodelling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2-deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in-depth analysis revealed that discrepancies in protamine ratios do not significantly influence the PTMs of histones in testicular sperm. In murine epididymal sperm, altered protamine ratios are associated with reduced acetylation of histone H4 (H4ac), a phenomenon similarly observed in ejaculated sperm from men. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2/PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodelling.

Protamine 2 ( Prm2/PRM2 ), together with Protamine 1 ( Prm1/PRM1 ), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodeling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2 -deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in-depth analysis revealed that discrepancies in protamine ratios do not significantly influence the post-translational modifications (PTMs) of histones in testicular sperm. In epididymal sperm these altered protamine ratios are associated with a reduction in the acetylation levels of histone H4 (H4ac), a phenomenon consistent across both murine and human samples. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2 / PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodeling.

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 ^-/- sperm, the short one (Mw 36 kDa) is mislocalized, while two long isoforms (Mw 40 and 41 kDa) are unexpectedly lost in Prm2 ^-/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/B2/B3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 protein or SUN4 expression in Prm2 ^-/- testes. Furthermore, the Prm2 ^-/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone– protamine 1/2 ratio and modified levels of cytoskeletal proteins. We detected retained Septin 12 isoforms (Mw 40 and 41 kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions, which was not true for healthy normozoospermic men. In conclusion, our findings expand the current knowledge regarding the connection between Protamine 2 and Septin 12 expression and localization, resulting in low sperm motility and morphological abnormalities.

Franchi and Tomsic (2023) correctly note that our review (Kämmerer et al., 2023) “has a clear focus on the technical aspect of RT-PCR, which is only one piece of the COVID-19 puzzle” and they ask for a more comprehensive discussion beyond our focus on the laboratory assay. They point to the lack of a specific definition of COVID-19 disease and conclude that, in order to test the first and the second of Koch’s postulates, there must be both a purified germ and a specified disease, neither of which was available for COVID-19. In reply, we address two questions they did not ask: 1. Are clinical symptoms induced by SARS-CoV-2 corroborated by RT-PCR? 2. Are Koch’s postulates valid for viruses? We assert that testing asymptomatic people is useless, whereas testing patients with clinical symptoms for a respiratory disease may enable a physician to confirm or reject a suspected diagnosis. Determining a diagnosis for any given patient is the physician’s challenge, while the researcher is responsible to show that the available tools are as near optimal as possible and to clarify the limitations of any such tools. Because there are no tools suitable for comprehensive and exclusive detection of infectious pathogens, we need to proceed carefully in applying the limited tools that do exist for tracing and tracking viral pathogens, to avoid under- or over-estimating a real or suspected pandemic.

For the first time in medical history, a laboratory assay (RT-PCR) was used as the sole criterion to diagnose a disease (COVID-19) and to define infectivity of a virus (SARS-CoV-2) without rating clinical symptoms and proof of replication-competent virus to justify implementing population-wide, untested interventions. The aims here are (1) to evaluate a robust quantitative RT-PCR (RT-qPCR) protocol that overcomes major concerns raised within the scientific community on the first WHO-recommended RT-qPCR protocol for SARS-CoV-2 sequences, (2) to characterize individual SARS-CoV-2 strains circulating in the Czech Republic from autumn 2020 to spring 2021 applying next generation sequencing and (3) to re-initiate scientific dialogue and return to reason and evidence-based medicine. We present a RT-qPCR test designed for the detection of all SARS-CoV-2 variants known so far without producing false-positives. Based on the genomic mutation profile, we demonstrate that the three individual waves (autumn 2020 to spring 2021) in the Czech Republic were successive, but lacked direct genomic relationship between each other. This became obvious with the omicron variant, which did not reveal direct evolutionary connection to any of the previous SARS-CoV-2 variants. In addition, we provide evidence that neglected principles of good scientific practice resulted not only in the publication of the WHO-recommended Charité RT-qPCR protocol, but also in health-related problems. Unnecessary quarantine of healthy individuals, as well as lockdowns and atrocious collateral damage on societies and economies worldwide due to a high number of false-positive “PCR-cases.” Otherwise, infectious symptomatic individuals were given a false sense of security by false-negative test results, which could lead to COVID-19 clusters. Both our results and literature data confirm that validation of any PCR-based diagnostic test by sequencing is mandatory on a regular basis. To prevent future misconduct, science needs a reality check and must re-initiate the scientific dialogue and liberate itself from political influence and dogma.

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/− mice were subfertile, whereas Prm1−/− mice were infertile. Prm1−/− and Prm2−/− sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/− sperm displayed only moderate DNA damage. The majority of Prm1+/− sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/− sperm. However, sperm from Prm1+/− and Prm1−/− mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/− animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

Background: Chronic Prostatitis/Chronic Pelvic Pain Syndrome (CP/CPPS) is a frequent disease affecting men of every age and accounting for a great number of consultations at urology departments. Previous studies suggested a negative impact of CP/CPPS on fertility. As increasing attention has been attributed to additional aspects, such as sperm DNA integrity and sperm protein alterations, besides the WHO standard semen analysis when assessing male fertility, in this prospective study, we aimed to further characterize the fertility status in CP/CPPS patients with a focus on these parameters. Methods: Sperm DNA fragmentation measured by sperm chromatin structure assay (SCSA) and protamine 1 to protamine 2 mRNA ratio assessed by RT-qPCR were analyzed along with conventional ejaculate parameters and inflammatory markers in 41 CP/CPPS patients and 22 healthy volunteers. Results: We found significant differences between the groups concerning multiple conventional ejaculate parameters. A significant increase in sperm DNA fragmentation was shown in CP/CPPS patients with association to other sperm parameters. The majority of CP/CPPS patients exhibited protamine mRNA ratios out of the range of regular fertility. Conclusions: This is a pioneering study with a strong practical orientation revealing that CP/CPPS leads to increased sperm DNA damage and changes in sperm protamine levels, emphasizing an unfavorable impact of CP/CPPS on fertility.

The LINC (LInker of Nucleoskeleton and Cytoskeleton) complex is localized within the nuclear envelope and consists of SUN (Sad1/UNc84 homology domain-containing) proteins located in the inner nuclear membrane and KASH (Klarsicht/Anc1/Syne1 homology domain-containing) proteins located in the outer nuclear membrane, hence linking nuclear with cytoplasmic structures. While the nucleoplasm-facing side acts as a key player for correct pairing of homolog chromosomes and rapid chromosome movements during meiosis, the cytoplasm-facing side plays a pivotal role for sperm head development and proper acrosome formation during spermiogenesis. A further complex present in spermatozoa is involved in head-to-tail coupling. An intact LINC complex is crucial for the production of fertile sperm, as mutations in genes encoding for complex proteins are known to be associated with male subfertility in both mice and men. The present review provides a comprehensive overview on our current knowledge of LINC complex subtypes present in germ cells and its central role for male reproduction. Future studies on distinct LINC complex components are an absolute requirement to improve the diagnosis of idiopathic male factor infertility and the outcome of assisted reproduction.