Abstract
In brief
Protamines package and shield the paternal DNA in the sperm nucleus and have been studied in many mouse models over decades. This review recapitulates and updates our knowledge about protamines and reveals a surprising complexity in protamine function and their interactions with other sperm nuclear proteins.
Abstract
The packaging and safeguarding of paternal DNA in the sperm cell nucleus is a critical feature of proper sperm function. Histones cannot mediate the necessary hypercondensation and shielding of chromatin required for motility and transit through the reproductive tracts. Paternal chromatin is therefore reorganized and ultimately packaged by protamines. In most mammalian species, one protamine is present in mature sperm (PRM1). In rodents and primates among others, however, mature sperm contain a second protamine (PRM2). Unlike PRM1, PRM2 is cleaved at its N-terminal end. Although protamines have been studied for decades due to their role in chromatin hypercondensation and involvement in male infertility, key aspects of their function are still unclear. This review updates and integrates our knowledge of protamines and their function based on lessons learned from mouse models and starts to answer open questions. The combined insights from recent work reveal that indeed both protamines are crucial for the production of functional sperm and indicate that the two protamines perform distinct functions beyond simple DNA compaction. Loss of one allele of PRM1 leads to subfertility whereas heterozygous loss of PRM2 does not. Unprocessed PRM2 seems to play a distinct role related to the eviction of intermediate DNA-bound proteins and the incorporation of both protamines into chromatin. For PRM1, on the other hand, heterozygous loss leads to strongly reduced sperm motility as the main phenotype, indicating that PRM1 might be important for processes ensuring correct motility, apart from DNA compaction.
Introduction
Vertebrate reproduction relies on the successful transport of the paternal chromatin to the oocyte by motile sperm cells. The formation of functional sperm requires a unique and well-orchestrated differentiation cascade in the male gonads. Crucial for the successful transport of the paternal DNA is the elongation and condensation of the sperm cell nucleus into a hydrodynamic shape. The necessary size reduction and shielding of DNA required for motility and transit through the reproductive tracts cannot be achieved by histones. Thus, paternal chromatin is completely remodeled from nucleo-histone to nucleo-protamine during spermiogenesis. As a consequence, DNA in mammalian sperm is hypercondensed to an almost crystalline state, considerably reducing the size of nuclei and rendering mature sperm transcriptionally inactive (Chandley & Kofman-Alfaro 1971, Sega 1974, Kierszenbaum & Tres 1975, Pogany et al. 1981, Balhorn 1982). While the sperm chromatin of most mammals is packaged by one protamine, primates and most rodents, as well as subset of other placental mammals express two protamines, protamine 1 (PRM1) and protamine 2 (PRM2).
Both genes are transcribed in round spermatids (steps 7–9 in mice) (Hecht et al. 1986a, b), after which mRNA is stored in the form of cytoplasmic ribonucleoprotein particles until translation in elongating spermatids (steps 12–16) (Kleene et al. 1984, Kleene 1989). Following transcript storage, murine Prm1 seems to be translated earlier (stage 12) than Prm2 (stage 14/15) (Klaus et al. 2016). PRM1 is a small protein (50 aa in mice), containing an arginine-rich DNA binding domain, flanked by short serine- and threonine-containing sequences (Fig. 1). PRM2 is larger (106 aa in mice) than PRM1, containing an arginine-rich DNA-binding domain and serine and threonine residues throughout its sequence (Balhorn 2007, Balhorn et al. 2018) (Fig. 1). Unlike PRM1, PRM2 is expressed as a precursor protein (Yelick et al. 1987). Upon DNA-binding, the N-terminal cleaved-PRM2 domain (cP2) is sequentially cleaved off, leaving only the mature-PRM2 (mP2) domain bound to DNA. Among placental mammals, both protamines contain numerous cysteine residues allowing for the formation of disulfide bridges, stabilizing the condensed chromatin (Balhorn et al. 1992) (Fig. 1). Additionally, PRM2 is able to bind one zinc atom per molecule, allowing for the formation of zinc bridges during epididymal maturation (Bench et al. 2000).
Amino acid alignments of human and mouse PRM1 and PRM2. PRM2 domains cP2 (cleaved-PRM2) and mP2 (mature-PRM2) and DNA binding regions are marked. Amino acids are colored red through blue according to their hydrophobicity value, where red is the most hydrophobic and blue is the most hydrophilic (Geneious Prime v 2019.2.3).
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Amino acid alignments of human and mouse PRM1 and PRM2. PRM2 domains cP2 (cleaved-PRM2) and mP2 (mature-PRM2) and DNA binding regions are marked. Amino acids are colored red through blue according to their hydrophobicity value, where red is the most hydrophobic and blue is the most hydrophilic (Geneious Prime v 2019.2.3).
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Amino acid alignments of human and mouse PRM1 and PRM2. PRM2 domains cP2 (cleaved-PRM2) and mP2 (mature-PRM2) and DNA binding regions are marked. Amino acids are colored red through blue according to their hydrophobicity value, where red is the most hydrophobic and blue is the most hydrophilic (Geneious Prime v 2019.2.3).
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Protamines have been discovered almost 150 years ago (Miescher 1874) and their importance for male fertility has been studied extensively. Several outstanding protamine reviews were published, containing detailed information of protamine evolution, biochemistry, function and relation to infertility (Oliva & Dixon 1991, Braun 2001, Oliva 2006, Balhorn 2007, Carrell et al. 2007, Rathke et al. 2014, Balhorn et al. 2018). We refer the reader to these excellent works for details or topics we cannot include here. This review aims to provide an updated summary of our knowledge of protamines and their function as learned from the analysis of different mouse models and related in vitro studies. We will specifically emphasize the differences between PRM1 and PRM2 function.
Box 1 Evolutionary history and selective pressures
Protamines are present in the sperm of both protostomes and deuterostomes (Oliva & Dixon 1991). They most likely developed in chordates from a sperm-specific histone H1 variant via a transition from a lysine-rich histone H1 to an arginine-rich protamine (Lewis et al. 2004). It is assumed that the selective restrictions imposed by internal fertilization have driven the evolution toward an arginine-rich protamine (Kasinsky et al. 2011). Because of its guanidinium group, arginine richness results in a stronger affinity for the protein to attach to the DNA molecule as well as better binding flexibility (Cheng et al. 2003, Ausio et al. 2014). Protamines in eutherian mammals evolved to be quite small compared to other species. Eutherian protamines, unlike other species, include cysteine residues, which allow the formation of complex tertiary structures by forming disulfide bridges between and among protamines (Balhorn 1982, Oliva & Dixon 1991, Balhorn et al. 1995).
Due to their involvement in reproduction, proteins of the male and female germline are often the target of selective pressures driven by sexual selection or male–female coevolution (Swanson & Vacquier 2002, Turner et al. 2008). Comparative evolutionary studies can reveal these pressures allowing us to draw conclusions about the function and importance of reproductive genes and proteins by evaluating the naturally occurring phenotypic diversity and associating it to changes and selective pressures in gene sequences and expression rates. When compared to other sperm nuclear basic proteins, some studies suggest that protamines are the quickest evolving reproductive proteins, with immense structural heterogeneity (Oliva & Dixon 1991, Wyckoff et al. 2000). For Prm1, however, evidence of purifying selection (i.e. selection against detrimental mutations in the coding sequence) focusing on the maintenance of the high arginine content exists. The location of arginine residues appears to be variable, indicating that Prm1 may be impacted by both positive (i.e. selection favoring beneficial mutations in the coding sequence) and purifying selection (Rooney et al. 2000, Lüke et al. 2016a ). Notably, it was found that the arginine content of PRM1 correlated with sperm head size and was maintained by sexual selection across mammals (Lüke et al. 2016a ).
Prm2 on the other hand seems to evolve under less-selective constraints. Studies including primates and rodents, as well as other Prm2 expressing mammalian species surprisingly indicate that the two domains of Prm2 (cP2 and mP2) seem to evolve under different selective pressures. The cP2 domain was shown to be highly conserved, whereas the mP2 domain was found to evolve at a faster rate indicating positive selection in primates and relaxed selective constraint in rodents (Lüke et al. 2011). Interestingly in rodents, changes in the cP2 coding sequence are significantly correlated to sperm head width and elongation (Lüke et al. 2014a). A potential reason for these different selection patterns is that cP2 function needs to be conserved, while mP2, putatively being redundant to PRM1 is free to evolve under less constraint (Lüke et al. 2011, 2016a). Since the cleaving of PRM2 seems to takes place while associated with chromatin and is achieved over a period of several days (Carré-Eusèbe et al. 1991, Chauvière et al. 1992), DNA condensation coincides temporally with PRM2 cleaving and posttranslational processing (Kierszenbaum & Tres 1975, Lee et al. 1995, Brewer et al. 2002). Based on this, Lüke et al. proposed that the cP2 domain may regulate the dynamics of DNA condensation, while the mP2 domain is mainly involved in the maintenance of the hypercondensed state of the mature sperm nucleus (Lüke et al. 2016a, b).
The histone to protamine transition
Chromatin remodeling is preceded by extensive histone modification, especially histone H4 acetylation, orchestrated by the interaction between histone acetyltransferases and histone deacetylases (Candido & Dixon 1972, Meistrich et al. 1992, Hazzouri et al. 2000, Marcon & Boissonneault 2004, Shirakata et al. 2014). Further, canonical histones are replaced by testis-specific variants, such as the histone H1 variant HILS (Yan et al. 2003), the histone H2B variant TH2B (Montellier et al. 2013, Shinagawa et al. 2015) and the histone H2A variant H2A.L1/2 (Govin et al. 2007, Barral et al. 2017). Chromatin remodeling was shown to be aided by various proteins, complexes and chaperones. During the process of chromatin reorganization, topoisomerase II, for example, induces double-strand breaks, that are believed to relieve supercoiled DNA strands and are subsequently repaired (McPherson & Longo 1993, Marcon & Boissonneault 2004, Laberge & Boissonneault 2005). We refer the reader to the excellent review by Rathke et al. for an overview of proteins involved in the processes preceding protamine binding (Rathke et al. 2014). We will concentrate here on proteins that are known or believed to be directly involved with protamine loading, during the final steps of spermiogenesis (e.g., CAMK4, BRDT, TH2B, H2A.L.2 and transition proteins (TNPs)) (Fig. 2). Barral et al. demonstrated that the testis-specific histone variant H2A.L.2, in collaboration with TH2B and TNPs (TNP1 and TNP2), induces structural alterations in chromatin that allow protamines to bind DNA (Barral et al. 2017). Protamines themselves then replace histones and histone–TNP complexes are evicted (Barral et al. 2017). Phosphorylation of protamines was shown to be crucial for correct protamine binding (Pirhonen et al. 1994a, b, Wu et al. 2000, Papoutsopoulou et al. 1999). Once bound to DNA, murine protamines seem to be dephosphorylated thereby ‘locking’ them in place (Fig. 2).
Schematic representation of the histone-to protamine-transition based on current knowledge from mouse models and in vitro studies as discussed in this review. PRM1 in green and PRM2 in yellow.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Schematic representation of the histone-to protamine-transition based on current knowledge from mouse models and in vitro studies as discussed in this review. PRM1 in green and PRM2 in yellow.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Schematic representation of the histone-to protamine-transition based on current knowledge from mouse models and in vitro studies as discussed in this review. PRM1 in green and PRM2 in yellow.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
The binding of protamines results in the coiling of DNA into a toroidal structure (Ward & Coffey 1991). The current consensus model of protamine-mediated chromatin condensation states that one protamine molecule binds to each turn of DNA in the major groove, neutralizing the phosphodiester backbone charge and causing the DNA to coil into toroidal structures (Ward & Coffey 1991, Balhorn 2007, Cree et al. 2011). After histone and TNP eviction is completed, during epididymal transit, the protamine–DNA complex is further stabilized by inter- and intra-protamine disulfide bonds and zinc bridges (Calvin & Bedford 1971, Balhorn 1982, Oliva & Dixon 1991, Balhorn et al. 1995, Hutchison et al. 2017) (Fig. 2). The reorganization ultimately achieves a nearly complete silencing of transcription and efficient protection of the paternal chromatin (Oliva & Dixon 1991, Steger 1999).
Box 2 Histone retention
Even though chromatin is reorganized and histones are replaced by protamines, a small fraction of the genome remains packed by histones (1–10% in mice, 5–15% in men), conveying epigenetic information (Balhorn et al. 1977, Hammoud et al. 2009a, 2010, Lesch & Page 2014, Samans et al. 2014, Jung et al. 2017, Yamaguchi et al. 2018). Both canonical and testis-specific histone variants such as TH2B and H2A.L.2 have been shown to be retained in fully condensed sperm chromatin (Kim et al. 1987, Govin et al. 2007, Barral et al. 2017). Studies in human and mouse sperm reported histone retention to be biased toward promoters of genes expressed during early development (Gardiner-Garden et al. 1998, Arpanahi et al. 2009, Hammoud et al. 2009a, Brykczynska et al. 2010, Erkek et al. 2013, Lesch & Page 2014). Other works report that histone retention is enriched in repeat elements such as long and short interspersed nuclear elements or pericentric repeats (Pittoggi et al. 1999, Govin et al. 2007). In addition, histone retention was reported to be particularly concentrated on pericentric heterochromatin, which was proposed to be due to the ability of H2A.L.2 to interact with RNA in these chromatin regions (Hoghoughi et al. 2020). The methodological difficulty of separating protamine and histone-bound fractions from hypercondensed DNA might be the cause of the discrepancies between studies (Carone et al. 2014, Yamaguchi et al. 2018). Recent studies started to use the protamine chaperone nucleoplasmin for selective protamine removal prior to ChIPseq. Using this method, it was shown that H3K4me3 seems to be preferentially retained in CpG-rich promoters and H3K9me3 in satellite repeats (Yamaguchi et al. 2018). Overall, several studies were able to confirm the association of H3K4me3 to CpG-rich promoters of developmentally relevant genes (Hammoud et al. 2009a, Brykczynska et al. 2010, Erkek et al. 2013, Xu et al. 2018, Yamaguchi et al. 2018). Additionally, Lesch and Page described the presence of bivalent histone marks in histones retained around the promoter of developmentally important genes (Lesch & Page 2014). They propose that this epigenetic information may play a role in the preparation for totipotency of the zygote. Moreover, histone retention seems to be involved in maintaining epigenetic information in and around imprinted genes (Brykczynska et al. 2010, Hammoud et al. 2010, 2011, Erkek et al. 2013). How histone retention is regulated during chromatin reorganization is a matter of debate. Acetylation and butyrylation of H4 tails were reported to balance histone retention and removal. According to this model, the binding of BRDT to acetylated H4 allows for the removal of histones by TNPs, while butyrylated H4 escapes replacement (Goudarzi et al. 2014). On the other hand, the selective incorporation of specific histone variants, such as H2A.L.2, which creates an open nucleosome allowing for TNP loading, might determine the histone retention pattern (Govin et al. 2007, Barral et al. 2017). What seems clear is that synchronized histone removal and selective retention involve complex processes and several independent factors (Goudarzi et al. 2014).
The protamine ratio
The essentiality of both protamines for sperm chromatin rearrangement is evident from the extensive investigation using mouse models (Cho et al. 2001, 2003, Mashiko et al. 2013, Takeda et al. 2016, Schneider et al. 2020, Arévalo et al. 2022, Merges et al. 2022). However, it is still unclear why two protamines are required in some species whereas one protamine (PRM1) appears to be sufficient in others. Part of the answer might lie in studying the interaction between protamines and the protamine ratio in detail.
Exactly how protamines interact is purely based on theoretical models (Corzett et al. 2002, Cree et al. 2011). It is however very clear that maintaining the exact ratio between PRM1 and PRM2 expression and abundance in mature sperm is a key factor of proper sperm function (Balhorn et al. 1988, Cho et al. 2001, Steger et al. 2001, 2003, Aoki et al. 2005, Oliva 2006). Importantly, the protamine ratio (PRM1:PRM2) varies widely between mammalian species (0–70% PRM2) while it is almost constant within the same species (Corzett et al. 2002). In men, PRM1 and PRM2 seem to be proportioned almost equally, while PRM2 is slightly higher abundant in mice accounting for 65% of total protamine (Corzett et al. 2002). The standard procedure for the evaluation of the protamine ratio is based on acid-urea gel electrophoresis band density measurements or quantitative RT-PCR in case of measuring the protamine expression ratio. It should be noted, that the gel-based method has certain limitations, such as potential differences in staining, as well as the difficulty in discerning between different proteoforms. In line with this, Soler-Ventura et al. recently showed that the human protamine ratio measured by LC−MS intensities is significantly higher (1.76 ± 0.16) than reported in previous studies using gel-based assays (1.04 ± 0.16) (Soler-Ventura et al. 2020). This certainly warrants further investigation in different species.
Even though the cause and specific implications are still not understood, changes in species-specific protamine ratios can disrupt sperm development and seem to be associated with male infertility even more so than absolute protamine amounts in humans (Balhorn et al. 1988, Steger et al. 2001, 2003). Modifications in the protamine ratio are associated with subfertility and increased DNA fragmentation in humans (Aoki et al. 2005, Oliva 2006) (see also: Box 3). Consistent with these findings, comparative analyses including different rodent species suggest the protamine ratio to be of great importance for sperm competitiveness and the species-specific sperm head phenotype (Lüke et al. 2014b, Arévalo et al. 2021). Earlier studies indicate that species lacking PRM2 or producing very little PRM2 relative to PRM1 show stronger chromatin condensation and decondense DNA more slowly after fertilization (Perreault et al. 1988, Corzett et al. 2002, Gosálvez et al. 2011). A recent study showed that the protamine expression ratio seems to adapt toward a 1:1 proportion under the selective pressure of sperm competition in different rodent clades (Arévalo et al. 2021) (see also: Box 1). If sperm competition-driven selection prefers a clear optimum, other selective factors should be at work to produce such different protamine ratios between species. Aside from condensing the nucleus and shielding paternal DNA, protamine expression levels and the protamine expression ratio may be related to the level of histone retention. And in fact, there appears to be an inverse relationship between the protamine expression ratio and the degree of histone retention in sperm chromatin in men (Hammoud et al. 2009b). Sperm histone retention (Box 2) is important for early embryonic development and potentially for the preservation of parental imprinting (Brykczynska et al. 2010, Hammoud et al. 2011, Yamaguchi et al. 2018). Under these terms, the protamine ratio might reflect a complex interplay between protamines and histones to organize and maintain the correct, species-specific histone retention pattern.
Lessons learned from mouse models
The fact that rodent and primate sperm express two protamines, that show considerable differences in sequence composition and processing, raises four main questions:
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Are both protamines required for the production of functional sperm?
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Do they perform different functions during chromatin condensation?
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What is the role of PRM2 processing?
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Do protamines interact with TNPs and other proteins involved in the histone-to-protamine transition?
Protamine-deficient mouse models
Over the last two decades, several protamine-deficient mouse models have been generated using a variety of different approaches (Cho et al. 2001, 2003, Mashiko et al. 2013, Takeda et al. 2016, Schneider et al. 2020, Arévalo et al. 2022, Merges et al. 2022) (Table 1).
Overview of mouse models discussed in this review.
| Gene/study | Genotype | Fertility | Phenotype |
|---|---|---|---|
| Prm1 | |||
| Cho et al. (2001) | Chimera | Infertile | Presence of unprocessed PRM2 |
| Mashiko et al. (2013) | Prm1+/− | Infertile | Fertilization incompetent sperm, reduced motility |
| Takeda et al. (2016) | Prm1+/− | Infertile | Fertilization incompetent sperm (fertilization possible when using IVF with zona-free oocytes) |
| Moritz et al. (2021) | Prm1K49A/K49A | Subfertile | Presence of unprocessed PRM2, increased histone retention, reduced motility |
| Merges et al. (2022) | Prm1+/− | Subfertile | Presence of unprocessed PRM2, impaired protamination, reduced motility |
| Merges et al. (2022) | Prm1−/− | Infertile | Inviable sperm, DNA degradation, increased histone retention, presence of unprocessed PRM2 |
| Prm2 | |||
| Cho et al. (2001, 2003) | chimera | Infertile | Impaired DNA condensation, presence of unprocessed PRM2 |
| Schneider et al. (2016, 2020) | Prm2+/− | Fertile | |
| Schneider et al. (2016, 2020) | Prm2−/− | Infertile | Inviable sperm, DNA degradation, increased histone retention |
| Itoh et al. (2019) | Prm2S56A/S56A | Fertile | |
| Arévalo et al. (2022) | Prm2+/Δc | Infertile | Inviable sperm, DNA degradation, TNP retention, failed H2A.L.2 retention, reduced protamine content |
| Arévalo et al. (2022) | Prm2-/Δc | Infertile | Inviable sperm, DNA degradation, TNP retention, failed H2A.L.2 retention, reduced protamine content |
| Brdt | |||
| Shang et al. (2007) | BrdtΔBD1/ΔBD1 | Infertile | Abnormal sperm, increased histone retention |
| Gaucher et al. (2012) | BrdtΔBD1/ΔBD1 | Infertile | Failed TNP and protamine incorporation, increased histone retention |
| Th2b | |||
| Montellier et al. (2013) | Th2b−/− | Fertile | |
| Montellier et al. (2013) | Th2b+/tag | Infertile | Developmental arrest at condensing spermatid stage |
| H2A.L.2 | |||
| Barral et al. (2017) | H2A.L.2−/− | Infertile | Impaired TNP incorporation, presence of unprocessed PRM2 |
| Tnp1 | |||
| Yu et al. (2000) | Tnp1−/− | Subfertile | Incomplete DNA condensation, presence of unprocessed PRM2, altered protamine ratio |
| Tnp2 | |||
| Yu et al. (2000) | Tnp2−/− | Subfertile | Incomplete DNA condensation, presence of unprocessed PRM2 |
| Tnp1/Tnp2 | |||
| Shirley et al. (2004) | Tnp1+/−/Tnp2+/− | Fertile | |
| Zhao et al. (2004) | Tnp1−/−/Tnp2−/− | Infertile | Defects in testicular sperm release, aberrant DNA condensation, presence of unprocessed PRM2 |
| Camk4 | |||
| Wu et al. (2000) | Camk4−/− | Infertile | Failed PRM2 deposition, TNP retention |
| nGpx4 | |||
| Pfeifer et al. (2001) | nGPx4−/− | Fertile | Delayed nuclear compaction |
| Conrad et al. (2005) | nGPx4−/− | Fertile | |
| Puglisi et al. (2014) | nGPx4−/− | Fertile | Interaction with prms |
First, Cho et al. deleted Prm1 and Prm2 coding regions by classical gene targeting, i.e. the transfection of a construct encoding for PGK-neo cassette flanked by DNA sequences homologous to the Prm1 and Prm2 sequence (Cho et al. 2001, 2003). Using homologous recombination, they generated Prm1+/− and Prm2+/− embryonic stem (ES) cells, which upon blastocyst injection produced several male and female chimeras. Interestingly, none of the chimeras were able to generate ES-derived offspring. However, ES cells seemed to have contributed to the development of the germ line, since sperm derived from the ES cells was detected. Cho et al. reported that chimeras with higher amounts of ES-derived sperm displayed more morphologically abnormal sperm with lower chromatin integrity. The researchers concluded that the DNA in the protamine deficient ES cell-derived sperm was incompletely condensed and therefore labile or degraded. Chimeric Prm1 and Prm2 males with larger amounts of ES-derived sperm showed increased amounts of unprocessed PRM2 in mature sperm. Cho et al. showed that chromatin structure was altered in ES-derived sperm and hypothesized that this causes the defects in PRM2 processing. The authors concluded that haploinsufficiency of PRM1 and PRM2 renders male mice infertile (Cho et al. 2001).
More than a decade later, however, Schneider et al. were able to establish a PRM2-deficient mouse line (Schneider et al. 2016). Contradicting the earlier results based on PRM2-deficient chimeras (Cho et al. 2001, 2003), Prm2+/− mice were reported to be fertile (Schneider et al. 2016). Schneider et al. hypothesized that expression of the neo-cassette in the mouse model published by Cho et al. resulted in trans-silencing of the nearby Prm1 gene (Schneider et al. 2016). In fact, such trans silencing had been described by Olson et al. who demonstrated that depending on the orientation of the PGK-neo cassette inserted to disrupt the MRF4 locus the neighboring gene Myf5 was silenced as well (Olson et al. 1996). In the study by Cho et al., sperm of chimeric PRM2-deficient mice showed a significant reduction in PRM1 level supporting the hypothesis of trans-silencing (Cho et al. 2001, 2003). Since Schneider et al. reported Prm2+/− males to be fertile, Prm2−/− mice could be generated and analyzed.
Prm2−/− mice are infertile (Schneider et al. 2016). Observed defects in DNA hypercondensation and increased DNA fragmentation correlate well with the described function of protamines and data from infertile men with impaired protamination. Additionally, PRM2-deficient murine sperm displayed disintegrated membranes, decreased sperm head size and viability as well as acrosomal defects (Schneider et al. 2016, 2020) (Fig. 3). Additionally, Prm2−/− sperm were completely immotile. This is however clearly due to the fact that the sperm cells are inviable and degrading. Similar morphological malformations were reported for Prm2 chimeric animals (Cho et al. 2001, 2003) and in patients with abnormal protamine loading (Depa-Martynow et al. 2012, Rogenhofer et al. 2013, Iranpour 2014). Since these observed defects do not seem to be directly related to protamine function, they are most likely secondary defects (Schneider et al. 2016). Later studies revealed that Prm2−/− mature sperm showed a significant increase in histone retention (Arévalo et al. 2022, Merges et al. 2022). Proteomic profiling of PRM2-deficient sperm revealed a strong decline in reactive oxygen species (ROS) scavenger proteins SOD1 (superoxide dismutase 1) and PRDX5 (peroxiredoxin 5) (Schneider et al. 2020). Well-balanced ROS levels are essential for fertility, as low levels are required for sperm capacitation, acrosome reaction and sperm maturation. In contrast, overshooting levels of ROS result in oxidative DNA damage, lipid peroxidation and apoptosis (Tremellen 2008, Aitken et al. 2012). Thus, lower levels of SOD1 and PRDX5 result in the rise of ROS levels, causing DNA fragmentation, lipid peroxidation and the downstream secondary effects seen in PRM2-deficient mice (Schneider et al. 2020). These observations support the hypothesis that the degree of protamination might serve as a proxy for chromatin integrity assessed during epididymal transit as discussed previously (Carrell et al. 2007, Steger & Balhorn 2018). According to this hypothesis, faulty DNA protamination initiates a self-destruction cascade of sperm. This process seems to be initiated during epididymal transit in these mice since oxidative DNA damage and impaired acrosome status were not observed in PRM2-deficient testicular spermatids (Schneider et al. 2020). This opens up new possibilities for treatment regimens for infertile men if the proposed molecular processes can be transferred from the mouse model to the human situation.
Schematic summary of phenotypic effects in different protamine mouse models in relation to the observed changes in the protamine ratio. Black blocks indicate the average level of the measured parameter for the different genotypes. Fertility indicates a summary of the level of fertility from infertile to fertile taken from measures of average litter size and pregnancy rate of the genotype. DNA fragmentation indicates the average percentage of mature sperm found to have fragmented DNA according to transmission electron microscopy, agarose gel electrophoreses analysis and oxidative DNA damage marker staining of the genotype. Sperm motility indicates the average percentage of motile mature sperm for each genotype. PRM2-precursor/TNP/Histone retention summarizes the amount of PRM2 precursor/TNP/histone (H3 and H4) found in epididymal sperm in the different genotypes compared to WT levels. Data were taken from the studies indicated.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Schematic summary of phenotypic effects in different protamine mouse models in relation to the observed changes in the protamine ratio. Black blocks indicate the average level of the measured parameter for the different genotypes. Fertility indicates a summary of the level of fertility from infertile to fertile taken from measures of average litter size and pregnancy rate of the genotype. DNA fragmentation indicates the average percentage of mature sperm found to have fragmented DNA according to transmission electron microscopy, agarose gel electrophoreses analysis and oxidative DNA damage marker staining of the genotype. Sperm motility indicates the average percentage of motile mature sperm for each genotype. PRM2-precursor/TNP/Histone retention summarizes the amount of PRM2 precursor/TNP/histone (H3 and H4) found in epididymal sperm in the different genotypes compared to WT levels. Data were taken from the studies indicated.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
Schematic summary of phenotypic effects in different protamine mouse models in relation to the observed changes in the protamine ratio. Black blocks indicate the average level of the measured parameter for the different genotypes. Fertility indicates a summary of the level of fertility from infertile to fertile taken from measures of average litter size and pregnancy rate of the genotype. DNA fragmentation indicates the average percentage of mature sperm found to have fragmented DNA according to transmission electron microscopy, agarose gel electrophoreses analysis and oxidative DNA damage marker staining of the genotype. Sperm motility indicates the average percentage of motile mature sperm for each genotype. PRM2-precursor/TNP/Histone retention summarizes the amount of PRM2 precursor/TNP/histone (H3 and H4) found in epididymal sperm in the different genotypes compared to WT levels. Data were taken from the studies indicated.
Citation: Reproduction 164, 3; 10.1530/REP-22-0107
In a CRISPR/Cas methodology-focused study, Mashiko et al. generated Prm1+/− mice, which were reported to be infertile (Mashiko et al. 2013). However, very limited fertility testing was performed in which Prm1+/− male founders were mated with superovulated females and oocytes were isolated. Formation of pronuclei, which was considered a successful fertilization event, was not detected for Prm1+/− sperm. The authors reported that sperm of Prm1+/− males show morphological abnormalities and reduced motility.
In order to overcome the infertility observed in male PRM1-deficient animals, Takeda et al. (2016) targeted Prm1 in TT2-XO ES cells and generated female chimeras which transmitted the Prm1 deletion to the germline. Prm1+/− males were infertile, with similar testis weight compared to WT, suggesting that sperm production was not affected (Takeda et al. 2016). Prm1+/− sperm showed reduced/defective protamination, unprocessed PRM2 in mature sperm, reduced DNA integrity and various head and tail deformities (Takeda et al. 2016). Tail malformations were linked to disorganization of microtubules in the sperm tail and a reduced mitochondrial membrane potential resulting in a severe reduction of sperm motility. Further, Prm1+/− sperm showed accelerated acrosome reactions, which might contribute to the reduction in motility. Comparison of the expression profiles of Prm1+/− to WT cauda epididymal sperm revealed only little differences, indicating that transcriptional silencing was not disturbed in Prm1+/− sperm. Surprisingly, however, in vitro fertilization (IVF) of zona-free oocytes with sperm from Prm1+/− males generated offspring, suggesting that infertility of Prm1+/− males was rather caused by secondary effects on acrosome integrity and sperm motility.
Contrary to previous studies (Cho et al. 2001, Mashiko et al. 2013, Takeda et al. 2016), a recent study by Merges et al. generating PRM1-deficient mice by CRISPR/Cas9-mediated gene editing showed that Prm1+/− males are able to produce offspring by natural breeding (Merges et al. 2022). Prm1+/− males were, however, subfertile showing reduced average litter sizes and pregnancy frequencies. In Prm1+/− epididymal sperm, minor ROS-mediated DNA damages were detected. Epididymal sperm appeared condensed and only slight DNA fragmentation was detected. Confirming results from previous studies, Prm1+/− sperm showed a severe reduction in motility. Additionally, Merges et al. showed that the protamine ratio was shifted in Prm1+/− sperm (from 1:2 to 1:5). In accordance with the previously reported mouse models, protamination of mature sperm seemed to be impaired and unprocessed PRM2 was abundantly detected (Takeda et al. 2016, Merges et al. 2022) (Fig. 3).
The subfertility of Prm1+/− males allowed for breeding of Prm1−/− males, which were infertile. Testis weight and morphology, as well as the number of sperm produced, were comparable in Prm1−/−, Prm1+/− and WT mice. In Prm1−/− testis, however, reactive oxygen species (ROS)-mediated DNA damages were detectable from stage 15–16 testicular spermatids, earlier than reported for Prm2−/− mice by Schneider et al. (2016, 2020). Prm1−/− sperm damage intensified during epididymal transit, and the majority of DNA isolated from Prm1−/− cauda epididymal sperm was fragmented. Prm1−/− cauda epididymal sperm head shapes were altered, being generally smaller and thinner compared to Prm1+/− and WT sperm (Fig. 3). Additionally, Prm1−/− epididymal sperm showed disrupted membranes, damaged acrosomes and were therefore completely immotile. Further, similar to Prm2−/− males, histone retention was increased in Prm1−/− mature sperm and slight TNP retention was detected (Arévalo et al. 2022, Merges et al. 2022) (Fig. 3).
These recently established protamine mouse lines will allow for the generation of Prm1+/− Prm2+/− males. In principle, sperm of mice, lacking one allele of Prm1 and Prm2, would contain only 50% total amount of protamine but could be expected to display an unaltered protamine ratio. Such animals could answer the question of whether male mice remain fertile when the total amount of protamine is reduced, while the correct protamine ratio is maintained.
Protamine-modified mouse models
Great advancements have been made in describing the role of protamines and their function, yet the role of posttranslational processing and posttranslational modification (PTM) of protamines is just starting to be understood. Several recent studies based on mouse models have started to tackle these questions (Table 1).
A mouse model carrying a deletion of the cP2 domain has been recently established by Arévalo et al. addressing the role of PRM2 processing (Arévalo et al. 2022). Due to a targeted deletion of the cP2 domain, Prm2+/Δc mice express only mP2 from the gene-edited allele in addition to full Prm2 from the WT allele. Surprisingly, in contrast to Prm2+/−, male Prm2+/Δc mice were infertile. In order to test if mice expressing only Prm2Δc would be fertile, Prm2−/Δc mice were generated. However, Prm2−/Δc males were also infertile, demonstrating that the infertility of Prm2+/Δc males was not solely caused by an aberrant interaction with full-length PRM2 expressed from the WT allele. Since Prm2+/− males were fertile, the infertility of Prm2−/Δc males originates from the lack of the cP2 domain, demonstrating its essential role in male fertility. DNA of sperm from Prm2+/Δc and Prm2−/Δc males was degraded starting in condensed testicular spermatids and PRM2 incorporation into the DNA was reduced during late spermiogenesis. Arévalo et al. hypothesize that this was caused either by the prevention of nuclear localization of PRM2 or secondary eviction from the nucleus. Using a cP2-specific antibody, it was shown that in WT, unprocessed PRM2 can be found in the cytoplasm of condensed spermatids and in residual bodies indicating that significant amounts of PRM2 are not processed in the WT (Fig. 2). cP2 staining was absent in the nuclei of condensed spermatids, while the mP2-specific signal was present in the nuclei. In Prm2+/Δc mice, however, PRM2 precursor was detected in the nuclei of condensed spermatids and in epididymal sperm (Arévalo et al. 2022). These results indicate that what is interpreted as ‘incomplete PRM2 processing’ in many of the protamine-related mouse lines might actually be failed PRM2 precursor eviction or PRM2 precursor retention. This warrants further testing.
In contrast to Prm2−/− mice, failure of transcriptional silencing was not apparent in cP2-deficient mice (Schneider et al. 2020). However, Pcgf5 (polycomb group ring finger 5) was shown to be highly expressed in cP2-deficient testes (Arévalo et al. 2022). Pcgf5 is part of the polycomb repressive complex 1 (PRC1) mediating heritable histone modifications (Piunti & Shilatifard 2021). The involvement of cP2 with PRC1 is not clear yet and merits further investigation.
Interestingly, loss of the cP2 domain lead to a strong alteration of the protamine ratio (2:1–5:1), showing a lower percentage of PRM2 (including precursors) compared to the WT. Additionally, the total amount of protamine is severely reduced in epididymal sperm. Thus, not only PRM2 but also PRM1 failed to correctly incorporate into chromatin in cP2-deficient mice. As mentioned earlier, large amounts of unprocessed PRM2 can be detected in mature sperm of PRM1-deficient mice (Merges et al. 2022). The PRM2 precursor therefore seems to be tightly connected to PRM1 incorporation, while the presence of PRM1 is a prerequisite for correct PRM2 processing or PRM2 precursor localization.
Remarkably, cP2-deficient epididymal sperm showed a strong retention of TNP1, but no generalized increase in histone retention. However, in cP2-deficient condensing spermatids, the histone variant H2A.L.2, which is critically involved in the histone-to-protamine transition, seemed to be localized in aberrantly shaped heterochromatin foci in the nucleus. In epididymal sperm of cP2-deficient males, H2A.L.2 was significantly lower abundant compared to WT. Thus, the cP2 domain seems to be directly involved in TNP removal and H2A.L.2 retention and is required for full sperm chromatin protamination (Arévalo et al. 2022) (Fig. 2). In line with these results, a recent study using a different cP2 antibody demonstrated that TNPs and the PRM2 precursor were consistently co-detected suggesting that TNPs and PRM2 do not act successively as previously assumed but simultaneously during histone-to-protamine transition (Rezaei-Gazik et al. 2022). In fact, Arévalo et al. were able to show that the cP2 domain tagged with eGFP was able to pull down untagged TNP1 when co-expressed in human embryonic kidney cells (HEK293). This suggests that cP2 might directly interact with TNP1 in vivo. Further, they proposed that the comparably low TNP retention found in PRM1-deficient sperm might be related to the strong presence of unprocessed PRM2, rather than a direct effect of PRM1 deficiency (Arévalo et al. 2022).
PTMs of histones like acetylation, methylation and phosphorylation are well-known epigenetic modifications that represent a histone code, which strongly affects chromatin structure and gene expression (Jenuwein & Allis 2001). PTMs have been also discovered on sperm histones as well as protamines. Phosphorylation was shown to be crucial for protamine DNA binding (Louie & Dixon 1972, Gusse et al. 1986, Pirhonen et al. 1994a, b). Brunner et al. discovered seven PTMs for murine PRM1 and four PTMs for murine PRM2, including serine and threonine phosphorylation, serine and lysine acetylation as well as lysine methylation marks (Brunner et al. 2014). A recent study by Soler-Ventura et al. provides a very detailed analysis of protamine PTMs and isoforms using a combined mass spectrometry-based approach. Remarkably, they were able to describe novel truncated PRM1 and unprocessed PRM2 proteoforms in humans, including a wide range of PTMs (Soler-Ventura et al. 2020). It is a matter of debate whether the sum of these modifications in protamines represents a protamine code which contributes to epigenetic intergenerational inheritance (Brunner et al. 2014, Castillo et al. 2015; reviewed in Blévec et al. 2020). The molecular function of these PTMs is largely unknown. Two recent studies revealed that dephosphorylation of PRM2 at serine 56 and acetylation of PRM1 at lysine 49 are essential for sperm maturation and function (Itoh et al. 2019, Moritz et al. 2021 (preprint)). Itoh et al. showed that serine 56 can be dephosphorylated by the testis- and sperm-specific phosphatase PPP1CC2. The phosphatase itself is a substrate of the HSP70S chaperone family. Presence of the co-chaperone HSPA4L, which acts as a nucleotide exchange factor, was proposed to support the release of PPP1CC2 from HSP70S, thereby allowing PPP1CC2 translocation to the nucleus and subsequently serine 56 dephosphorylation. Knockout of HSPA4L resulted in male infertility and impaired spermiogenesis with round-headed sperm, which were unable to initiate embryonic development following IVF or ICSI. Accordingly, the level of sperm chromatin-associated PPP1CC2 was decreased in HSPA4L-deficient sperm. However, the exchange of serine 56 to alanine (S56A) was able to rescue the phenotype of HSPA4L-deficient mice. Prm2S56A/S56A mice themselves were fertile and displayed no gross abnormalities. Thus, serine 56 and its PTM seem to be dispensable for fertility and sperm function. However, if present, the dephosphorylation of serine 56 is a prerequisite for sperm maturation during spermiogenesis (Itoh et al. 2019). Like murine protamines, human protamines undergo posttranslational phosphorylation and dephosphorylation (Pruslin et al. 1987, Chira et al. 1993). However, it remains to be investigated whether results from the mouse model can be associated with human male infertility as well.
In a recent study published as a preprint, Moritz et al. set out to investigate the significance of evolutionarily conserved non-arginine residues in protamine-DNA binding (Moritz et al. 2021 (preprint)). It is believed that the protamine-DNA binding dynamic relies primarily on passive electrostatic interaction with the arginine-rich binding domains (Gou et al. 2020). However, it was shown that PTMs of protamines, especially phosphorylation plays an additional role (Pirhonen et al. 1994b, Green 2001, Seligman et al. 2004). Moritz et al. found that these PTM sites seemed to be conserved within species but can vary between (Moritz et al. 2021 (preprint)). Focusing on PRM1 lysine 49 (P1 K49), which was found to be acetylated and conserved across rodents, mutant mice carrying a substitution of P1 K49 with alanine (K49A) were generated and shown to be subfertile in the homozygous state. While spermatogenesis and sperm count seemed to be unaffected, mature sperm show head morphology and midpiece morphological abnormalities and progressive motility of sperm seemed to be severely reduced. The mutation did not affect the levels of PRM1 and PRM2 or the protamine ratio, however unprocessed PRM2 was accumulated in mature sperm. Mutant sperm show histone, but no TNP retention. They concluded that PRM1 K49 acetylation might be necessary for the proper binding of PRM1 and successful eviction of nucleosomes. They hypothesize that the acetyl-lysine reader BRDT might be involved in assessing the lysine acetylation levels of protamines, including PRM1 K49 acetylation, and thereby in modulating the chromatin reorganization (Moritz et al. 2021 (preprint)).
Of note, the deletion of Prm1, Prm2 or the cP2 domain resulted in deviations from the WT protamine ratio of 1:2 (Schneider et al. 2016, 2020, Arévalo et al. 2022, Merges et al. 2022). In Fig. 3, we summarize the main phenotypes of the protamine mouse models described above in relation to the observed protamine ratio. Taken together, the phenotypes that were described for these different mouse models indicate that a shift in the ratio toward PRM2 might be more easily tolerated than a relative excess of PRM1 in mice (Fig. 3).
Protamine-interactor mouse models
During the post-meiotic phases, global chromatin hyperacetylation initiates the reorganization of paternal chromatin. The testis-specific bromodomain-containing protein BRDT not only plays an important role during the meiotic stages of spermatogenesis but is also involved in the initiation of chromatin hypercondensation (Fig. 2). BRDT’s bromo-domain 1 (BD1) reads acetyl-lysine marks and binds to acetylated histone H4. Thereby, it is proposed to drive the genome-wide replacement of histones with TNPs (Pivot-Pajot et al. 2003, Govin et al. 2004, Dhar et al. 2012). Different mouse models have been established to study the function of BRDT, but the deletion of BD1 specifically demonstrated its role during chromatin reorganization. In male mice lacking BD1 (Shang et al. 2007, Gaucher et al. 2012), both TNPs and protamines while detectable seem not to be incorporated into the chromatin of spermatids (Gaucher et al. 2012). Thus, BD1 seems to be instrumental for the eviction and replacement of histones.
During chromatin reorganization, canonical histones are replaced by testis-specific variants. Two of these variants, the histone H2B variant TH2B (Montellier et al. 2013, Shinagawa et al. 2015) and the histone H2A variant H2A.L.2 (Govin et al. 2007, Barral et al. 2017) play important roles during histone-to-protamine transition (Fig. 2). It was shown that H2A.L.2 and TH2B are prone to forming dimers (Govin et al. 2007).
TH2B, first described in 1975 (Branson et al. 1975, Shires et al. 1975), is expressed in meiotic spermatogonia where it gradually and completely replaces canonical H2B (Montellier et al. 2013). The role and function of TH2B were investigated using KO mouse models as well as a model in which TH2B was C-terminally tagged (Montellier et al. 2013). Strikingly, TH2B-deficient mice were fertile and other H2B variants were upregulated to compensate for the lack of TH2B. Although the loss of TH2B causes dramatic changes in core histone marks, it did not seem to affect litter sizes or health of the offspring. The mice harboring the C-terminally tagged TH2B on the other hand were infertile. In these mice, germ cell development was arrested in condensing spermatids, at a time when TNPs and protamines should have completely displaced TH2B. This suggests that correct interaction of TH2B, or compensatory H2B variants with H2A.L.2, TNPs and/or protamines is necessary during the final stages of spermiogenesis.
H2A.L.2, described by Govin et al., is expressed and incorporated into DNA during late spermiogenesis, at the time of histone replacement and detected at the same time as TNPs (Govin et al. 2007, Barral et al. 2017). H2A.L.2-deficient mice are infertile but showed normal testis histology and sperm count. TNPs and protamines seemed to be present at normal levels. However, H2A.L.2 deficiency leads to incomplete loading of TNPs and similar to TNP deficiency accumulation of PRM2 precursor. Co-immunoprecipitation analyses further support an interaction between H2A.L.2, TH2B, TNP2 and PRM2. Thus, they conclude that H2A.L.2 integration into chromatin initiates a series of transitional phases eventually leading to the incorporation of protamines and the hypercondensation of the paternal DNA.
TNPs are likely to closely interact with protamines during protamine loading and final histone eviction. They are arginine- and lysine-rich with a strong affinity to binding DNA (Kistler et al. 1975, Heidaran & Kistler 1987, Kleene & Flynn 1987, Brewer et al. 2002). The function of these proteins is still not completely clear. It is believed that TNPs cooperate with topoisomerase II, rendering the DNA more flexible (Akama et al. 1998, Lévesque et al. 1998, Singh & Rao 1988, Meistrich et al. 2003), condense chromatin (Baskaran & Rao 1990, Brewer et al. 2002) and stimulate the repair of DNA breaks (Caron et al. 2001). Both TNP1- and TNP2-deficient mouse models have been generated and analyzed (Yu et al. 2000). Tnp1−/− and Tnp2−/− male mice were subfertile. TNPs seem to be able to partly compensate for each other since in Tnp1−/− and Tnp2−/− males only slight sperm abnormalities were described (Yu et al. 2000, Zhao et al. 2001). Both, Tnp1−/− and Tnp2−/− males showed abnormal chromatin condensation starting in step 11 spermatids, forming condensed foci. Additionally, chromatin condensation was incomplete in epididymal Tnp2−/− sperm. While histone eviction was not disrupted in Tnp1−/− or Tnp2−/− sperm, unprocessed PRM2 was detected in mature sperm. In epididymal sperm of Tnp1−/− males, the relative protamine ratio was shifted to 1:3 (including unprocessed PRM2) but remains unchanged in Tnp2−/− sperm. Strikingly, in Tnp1−/− spermatids, PRM2 seemed to be translated prematurely in part. If this premature PRM2 translation represents a compensatory mechanism is still unclear.
Tnp1−/−Tnp2−/− double KO mice were infertile, with reduced epididymal sperm counts correlating with defects in testicular sperm release (Zhao et al. 2004). The majority of Tnp1−/−Tnp2−/− sperm were inviable and immotile showing a severe head and tail deformations and aberrant chromatin condensation. Again, histone removal was unaffected by TNP loss. Both protamines were effectively deposited, but large amounts of PRM2 precursor were detected in epididymal sperm. Haploinsufficiency of one TNP while the other is absent caused similar phenotypical aberrations as the double KO (Shirley et al. 2004). Mice heterozygous for both TNPs, on the other hand, were fertile showing no obvious sperm defects again demonstrating the compensation.
So, in general, TNPs are not required for the deposition of protamines but the loss of TNPs seems to be connected to the accumulation of unprocessed PRM2 in epididymal sperm. In fact, Barral et al. were able to show that TNP2 is able to pull down both processed and unprocessed PRM2 in vitro (Barral et al. 2017). Together with the results from cP2-deficient mice, this suggests that the PRM2 precursor is closely connected to transition protein turnover. Unprocessed PRM2 being necessary for transition protein eviction while the presence of correct levels of TNPs is needed to prevent the accumulation of PRM2 precursor in the nucleus. The same relationship can be observed between PRM1 presence/incorporation and the PRM2 precursor.
Ca2+/calmodulin-dependent protein kinase IV (CAMK4) is a serine/threonine protein kinase, which is expressed in spermatids and associated with chromatin and the nuclear matrix (Wu et al. 2000). CAMK4−/− mice were infertile with spermiogenesis being arrested during nuclear condensation (stage IV). The transcript levels of TNPs and protamines and translational timings were not affected in CAMK4-deficient sperm. However, while PRM1 was detected throughout spermiogenesis, PRM2 was undetectable from step 15 spermatids onward. TNP2 on the other hand showed prolonged retention. This is congruent with the results from PRM2 and cP2-deficient mice indicating that PRM2 is required for TNP eviction. Wu et al. showed that CAMK4 phosphorylates PRM2 (in vitro) during or after proteolytic processing. Thus, CAMK4 seems to be involved in PRM2 phosphorylation, proper PRM2 deposition and consequently TNP removal (Wu et al. 2000).
Following histone and TNP eviction, during epididymal maturation, the sperm chromatin complex is stabilized by disulfide bonds and zinc bridges (Calvin & Bedford 1971, Balhorn 1982, Oliva & Dixon 1991, Balhorn et al. 1995). The sperm nuclear isoform of glutathione peroxidase 4 (nGPx4) contains an N-terminal arginine-rich sequence, which mediates nuclear localization and chromatin binding (Pfeifer et al. 2001, Moreno et al. 2003). Pfeifer et al. (2001) proposed nGpx4 to exhibit protamine thiol peroxidase activity that aids in stabilizing the hypercondensed chromatin by cross-linking protamine cysteine residues (Fig. 2). Conrad et al. showed that nGPx4-deficient mice were fertile but exhibit a delay in the completion of epididymal nuclear compaction (Conrad et al. 2005). Thus, while nGPx4 did seem to be involved in cross-linking protamine disulfides, the lack of this variant was eventually compensated for, possibly by other glutathione peroxidases or canonical GPx4 (Conrad et al. 2005). Further analyses by Puglisi et al. revealed that sperm from nGPx4-deficient mice decondensed faster than WT sperm in vitro, further suggesting that nGPx4 is involved in sperm chromatin compaction. They later showed that nGPx4 localized to the nuclear matrix and seemed to directly interact with protamines (Puglisi et al. 2014).
Many of the main factors orchestrating the histone-to-protamine exchange have been identified and studied (Table 1). The detailed interaction dynamics and the role of these complexes in regulating histone retention and modification, however, are still unclear and require further investigation (Fig. 2).
Questions answered
The extensive study of protamines and their interactors over decades allowed us to at least partly answer the four key questions posed at the beginning of this chapter.
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Are both protamines required for the production of functional sperm?
Indeed, both protamines are required for the production of functional sperm in mice. Prm1−/− and Prm2−/− mice showed strongly aberrant sperm phenotypes and infertility. In both cases, DNA was fragmented and oxidative damage was strongly apparent rendering sperm inviable.
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Do they perform different functions during chromatin condensation?
The results gathered from the different protamine-deficient mouse models do indicate that PRM1 and PRM2 perform distinct functions during spermiogenesis. First, strong TNP retention was only observed in PRM2 mouse models, while none of the published PRM1 mouse models reported more than weak TNP retention. Thus PRM2, specifically its cleaved domain, seems to be instrumental in TNP eviction from DNA/chromatin of sperm. Additionally, loss of one allele of Prm1 leads to subfertility whereas loss of one allele of Prm2 did not, and strikingly Prm1+/− mice showed a significant reduction in sperm motility but only slight DNA damage. Considering that the translation of Prm1 precedes Prm2, different hypotheses on the roles of these proteins can be posted. First, PRM1 could act as a vanguard preparing the field for PRM2. If PRM1 levels are low, this does not happen properly, allowing for an PRM2 ‘excess’ which might cause further problems, such as binding to DNA in regions it should not. Second, PRM1 might have a unique yet unknown function other than binding and condensing DNA affecting sperm motility.
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What is the role of PRM2 processing?
While the role of PRM2 processing and the cP2 domain is still not completely clear, the picture that emerges from studying cP2-deficient mouse lines points toward a role in orchestrating the eviction of TNPs, possibly their transport out of the nucleus and the regulation of the H2A.L.2 nucleosome. Recent results indicate that not all PRM2 is processed and that the precursor itself might perform a different function than the processed form. Additionally, these results lead to the hypothesis that only PRM2 that is bound to DNA is processed while the PRM2 precursor engaged in other processes is not cleaved and remains in cytoplasm and residual bodies. PRM2 processing or PRM2 precursor localization seems to depend on the tightly regulated balance of various proteins (H2A.L.2, TNP1, TNP2, PRM1 and PRM2 itself).
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Do protamines interact with TNPs and other proteins involved in the histone-to-protamine transition?
Cell culture assays revealed that TNP2 was able to interact with processed and unprocessed PRM2 via the mP2 domain, but interestingly not the cP2 domain. Additionally, initial results indicated that the cP2 domain was able to interact with TNP1. nGPX4 was shown to directly interact with protamines during chromatin cross-linking. Further interactions remain to be elucidated.
Lessons learned from in vitro studies
The complexity of spermiogenesis and the difficulty of working with and disentangling the different components of the hypercondensed chromatin restricts the experimental approaches for a detailed analysis of protamine function in vivo. Therefore, in vitro-based methods are increasingly helpful. Key questions that can be answered in vitro are the following:
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Is an interaction with TNPs and testis-specific histones necessary for DNA condensation by protamines?
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Does DNA binding affinity and DNA condensation efficiency differ between PRM1 and processed and unprocessed PRM2?
Iuso et al. demonstrated in sheep fibroblasts that mouse PRM1 binds to DNA and condenses nuclei when expressed in vitro (Iuso et al. 2015). PRM1 accumulated as nuclear speckles, which grow in size until the nucleus is condensed into a stick-like shape. Of note, they were able to inject these spermatid-like (2N) nuclei into enucleated oocytes where the DNA underwent decondensation, maternal histones were incorporated and development to blastocyst stage was reported (Iuso et al. 2015, Czernik et al. 2016). The ability to condense somatic nuclei in vitro has since been shown for mouse PRM2 and the mP2 domain as well (Arévalo et al. 2022).
In their recent preprint, Moritz et al. extensively evaluated differences between mouse PRM1 and PRM2, as well as unprocessed PRM2 and mutated PRM1 (K49A) in DNA binding affinity and compaction efficiency in vitro using electrophoretic mobility shift assays and DNA curtain assays (Moritz et al. 2021 (preprint)). Their results indicated that the mutated PRM1 had a significantly lower DNA binding affinity. While WT PRM1 selectively bound mP2 rather than the unprocessed PRM2, the mutated PRM1 lost this selectivity. Additionally, they were able to show that mP2 had a higher DNA binding affinity and compacted DNA strands faster than unprocessed PRM2 in these assays. DNA binding affinity was similar between PRM1 and mP2, but mP2 compacted DNA significantly faster and to a denser state than PRM1. Jointly and in the WT ratio of 1:2, PRM1 and PRM2 bound DNA more efficiently. However, PRM1 together with unprocessed PRM2 showed a much lower binding affinity (Moritz et al. 2021 (preprint)). This argues for a role of cP2 in reducing the pace of DNA condensation. This could serve to minimize the DNA damage occurring due to torsional stress. PRM2 processing-based slowing down of the condensation rate could in principle allow the double-strand repair system more time to fix DNA damage.
Earlier studies, mimicking DNA compaction by salmon protamines and arginine-rich peptides in vitro, showed that the rate of DNA condensation depended on the amount of protamine binding to the chromatin (Brewer et al. 1999) and that the stability of toroid structures formed was dependent on the arginine content of the protamine or peptide used (Brewer et al. 1999, Balhorn et al. 2000). Additionally, in vitro experiments using Syrian hamster protamines determined that the condensation and decondensation rates of PRM1 and PRM2 were similar (Brewer et al. 2002). Addition of zinc, however, increased the DNA condensation rate of PRM2 nearly three-fold, while it did not influence PRM1-mediated DNA condensation. Additionally, Björndahl and Kvist showed that disruption of zinc salt bridges was easier than dissolving disulfide bridges, which could result in faster decondensation of paternal chromatin after fertilization (Bjorndahl & Kvist 2014). Thus, the presence of zinc bridges in species expressing both protamines could be an explanation for the differences in the extent of DNA compaction between species expressing PRM1 and PRM2 as opposed to PRM1 only (Gosálvez et al. 2011).
Questions answered
Although in vitro-based studies of protamines and the histone-to-protamine transition are still ongoing and have great potential for enhancing our knowledge of these processes in the future, the questions posed can be partly answered:
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Is an interaction with TNPs and testis-specific histones necessary for functional DNA condensation by protamines?
At least for mouse PRM1 in sheep fibroblasts the answer seems to be no, PRM1 does not need to interact with TNPs or sperm-specific histones in order to condense DNA into a state that is apt for inducing embryo development until the blastocyst stage. Although mouse PRM2 and mP2 are able to condense free DNA molecules and somatic cell chromatin it has not been shown yet if the DNA is sufficiently shielded and intact to support embryo development.
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Does DNA binding affinity and DNA condensation efficiency differ between PRM1 and processed and unprocessed PRM2?
Depending on the species and experimental conditions (presence of zinc) DNA binding affinity and condensation efficiency does seem to differ between protamines and processed or unprocessed PRM2. Processed PRM2 seemed to condense DNA faster than PRM1. Unprocessed PRM2 seemed to have a comparably lower binding affinity to DNA and condensed DNA at a slower pace compared to processed PRM2. Thus, the cP2 domain could be necessary for controlling the pace of DNA compaction. Interestingly, processed PRM2 seemed to also have a stronger affinity to binding PRM1. Considering that both PRM1 and PRM2 abundance is reduced in cP2-deficient sperm this could indicate that mP2 and PRM1 intercept each other before binding DNA. This would suggest that cP2 might be able to block premature interaction with PRM1, congruent with the hypothesis that PRM2 is only processed when bound to DNA.
Box 3 Protamines and infertility in humans
Protamines have been studied extensively in light of their involvement in human male sub- and infertility (Oliva 2006, Carrell et al. 2007). Aberrant sperm protamine content affected sperm motility, morphology and sperm number in men (Aoki et al. 2005). The presence of PRM2 precursor in epididymal sperm was associated with decreased DNA integrity and sperm dysfunction (Yebra 1998, Torregrosa et al. 2006). DNA damage, in turn, leads to decreased survival of offspring (Ruiz-López et al. 2010). Several studies have reported sub- or infertility related to SNPs in protamine sequences (Tanaka et al. 2003, Aoki et al. 2006a, Ravel et al. 2007, Gázquez et al. 2008, Imken et al. 2009, Tüttelmann et al. 2010, Jodar et al. 2011, Yu et al. 2012). Mutations or polymorphisms of protamines were reported to induce conformational changes and affect incorporation into chromatin (Jodar et al. 2011, He et al. 2012). A meta-analysis concluded that the two most commonly found SNPs (PRM1 rs2301365 (190C>A) and PRM2 rs1646022 (298 G>C)) were likely to play a role in human fertility problems, but that more research is needed to support the clear association with male infertility (Jiang et al. 2018). The SNP rs2301365 locates to the 5′-UTR of PRM1, potentially affecting the expression of PRM1 (Jodar et al. 2011, He et al. 2012, Jiang et al. 2015, 2018). However, the strongest association with sub- and infertility in men has been reported for deviations of the protamine ratio (Balhorn et al. 1988, Belokopytova et al. 1993, Yebra & Oliva 1993, Khara et al. 1997, Bench et al. 1998a, Yebra 1998, Carrell & Liu 2001, Mengual et al. 2003, Oliva 2006, Torregrosa et al. 2006, Rogenhofer et al. 2013, Francis et al. 2014, Ni et al. 2016). Hence, the protamine ratio has been constituted as a biomarker for male fertility (Steger et al. 2008, Ni et al. 2016). A meta-analysis confirmed the association between aberrant sperm protamine ratio and subfertility (Ni et al. 2016). In addition, it seemed that PRM2 deregulation rather than PRM1 deregulation was the main cause of alterations in the protamine ratio, while a lower protamine ratio was associated with stronger DNA damage than higher ratios (Yebra & Oliva 1993, Carrell & Liu 2001, Aoki et al. 2005). However, alteration in the protamine ratio of mature sperm is not necessarily related to SNPs in protamine sequences or regulatory regions, but can also be caused by a mutation in interactors and potentially altered zinc levels. These factors should be considered in patients with altered protamine expression levels. Additionally, conventional ART techniques like IVF or ICSI with epididymal sperm show lower success rates for patients with impaired protamination (Nasr-Esfahani et al. 2004, Aoki et al. 2006b, Rogenhofer et al. 2013). ICSI of oocytes using testicular sperm from Prm2−/− mice induced the development of blastocyst stage embryos (Schneider et al. 2020), suggesting that this technique is a promising alternative treatment approach for men with protamine-related alterations.
Perspectives
Protamines are unique proteins, explicitly dedicated to packaging and protecting the paternal chromatin on its way to the fertilization site. Extensive investigation over the course of decades not only confirmed their essentiality many times but also strikingly highlighted that the role and function of protamines go beyond the simple packaging and shielding of DNA. The mere existence of a second protamine (Protamine 2) that has to be expressed in a certain ratio to the ubiquitous protamine 1 points toward high complexity in interaction dynamics and chromatin regulation. We suggest that by continuing to investigate the differences in protamine 1 and protamine 2 function, the role of the protamine ratio and protamine 2 processing, we will be able to unravel the whole range of protamine function. Their tightly restricted expression pattern and function together with recent technological advances, such as CRISPR/Cas, allow for relatively easy establishment and analysis of animal models. However, the tight compaction of sperm DNA presents restrictions in investigating interactions and protein complexes involved in chromatin compaction on a molecular level. Here in vitro studies will be indispensable. Taking what we learn from mouse models and applying it in in vitro settings, which will again inform further in vivo studies, will allow us to get a comprehensive picture of sperm chromatin dynamics. Further, comparative evolutionary studies allowed for conclusions about the essentiality of protamines, their domains and the protamine ratio as well as their function by carefully evaluating the naturally occurring phenotypic diversity and associating it to changes and selective pressures in gene sequences and expression rates. Thus, evolutionary-comparative approaches can significantly aid in our efforts to fully comprehend protamines. We would like to emphasize that only a transdisciplinary scheme, including biomedical, medical, cell molecular, biochemical, biophysics and evolutionary approaches, will lead us to fully understand how paternal chromatin is reorganized and how the fundamental genetic and epigenetic information is maintained for the next generation.
Lessons learned
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PRM1 and PRM2 DNA binding dynamics differ.While in vitro PRM1 and PRM2 had similar DNA binding affinities, processed PRM2 condensed DNA at a faster pace. Unprocessed PRM2 had a lower binding affinity to DNA and condensed DNA at a lower rate. In mice, Prm1 translation precedes Prm2 translation, suggesting distinct roles of both protamines in DNA binding rather than competition for DNA binding sites. PRM2 is processed upon binding to DNA in vivo, potentially slowing down DNA condensation and/or avoiding premature protamine–protamine interactions.
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PRM1 and PRM2 are both essential for fertility in mice. Shifts in the protamine ratio and changes in the protamine coding sequence have been related to male sub- and infertility both in mice and men. In mice, heterozygous deletion of Prm1 caused sperm defects and subfertility. One allele of Prm2, on the other hand, was sufficient to retain normal fertility. Loss of either protamine or the cP2 domain, however, lead to severe sperm damages and infertility.
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PRM1 and PRM2 have common and distinct functions.Both protamines participate in sperm DNA condensation. Unprocessed PRM2, however, carrying essential information in its N-terminus, seems to be involved in TNP-to-protamine turnover and H2A.L.2 retention. The process of PRM2 cleaving on the other hand might be essential to slow down DNA condensation. PRM1 is translated prior to PRM2 and seems to not only bind and condense DNA but also execute an additional function essential for sperm motility.
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PRM1 and PRM2 perform their functions in a complex interacting network.Prior to protamine loading, the stage is prepared by replacing canonical histones with specialized variants and global histone acetylation. This allows for binding of TNPs and the formation of specialized nucleosome complexes (H2A.L.2-TH2B-TNP). Protamines then come in to replace these complexes and possibly to regulate their eviction and the retention of a subset of nucleosomes (see Fig. 2). Studies have shown that when any of these key components are missing, chromatin condensation is negatively affected.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Our work was supported by DFG grants to L A (AR 1221/1-1), H S (Scho 503/15-2; Scho 503/23-1) and S S (Schn 1668/1-1).
Author contribution statement
L A, G M, S S and H S wrote, discussed and edited the manuscript.
Acknowledgements
The authors are very grateful to their colleagues whose excellent research was included in this review, as well as to their colleagues for writing the outstanding reviews that were published before this one on similar topics. We apologize to colleagues whose publications are not referenced here, due to space limits. We thank the anonymous reviewers for their constructive criticism and excellent suggestions, which considerably improved this manuscript.
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