Protamines are the safeguards of the paternal sperm genome. They replace most of the histones during spermiogenesis, resulting in DNA hypercondensation, thereby protecting its genome from environmental noxa. Impaired protamination has been linked to male infertility in mice and humans in many studies. Apart from impaired DNA integrity, protamine-deficient human and murine sperm show multiple secondary effects, including decreased motility and aberrant head morphology. In this study, we use a Protamine-2 (Prm2)-deficient mouse model in combination with label-free quantitative proteomics to decipher the underlying molecular processes of these effects. We show that loss of the sperm’s antioxidant capacity, indicated by downregulation of key proteins like Superoxide dismutase type 1 (SOD1) and Peroxiredoxin 5 (PRDX5), ultimately initiates an oxidative stress-mediated destruction cascade during epididymal sperm maturation. This is confirmed by an increased level of 8-OHdG in epididymal sperm, a biomarker for oxidative stress-mediated DNA damage. Prm2-deficient testicular sperm are not affected and initiate the proper development of blastocyst stage preimplantation embryos in vitro upon intracytoplasmic sperm injection (ICSI) into oocytes. Our results provide new insight into the role of Prm2 and its downstream molecular effects on sperm function and present an important contribution to the investigation of new treatment regimens for infertile men with impaired protamination.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma‐like TCam‐2 cells to SOX17 in somatic cells and SOX2 in EC‐like 2102EP cells. In seminoma‐like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17‐binding sites overlap in seminoma‐like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma‐like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC‐like cells SOX2 regulates pluripotency‐associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.

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.

Profilins (PFNs) are key regulatory proteins for the actin polymerization in cells and are encoded in mouse and humans by four Pfn genes. PFNs are involved in cell mobility, cell growth, neurogenesis, and metastasis of tumor cells. The testes-specific PFN3 is localized in the acroplaxome–manchette complex of developing spermatozoa. We demonstrate that PFN3 further localizes in the Golgi complex and proacrosomal vesicles during spermiogenesis, suggesting a role in vesicle transport for acrosome formation. Using CRISPR/Cas9 genome editing, we generated mice deficient for Pfn3. Pfn3–/– males are subfertile, displaying a type II globozoospermia. We revealed that Pfn3–/– sperm display abnormal manchette development leading to an amorphous sperm head shape. Additionally, Pfn3–/– sperm showed reduced sperm motility resulting from flagellum deformities. We show that acrosome biogenesis is impaired starting from the Golgi phase, and mature sperm seems to suffer from a cytoplasm removal defect. An RNA-seq analysis revealed an upregulation of Trim27 and downregulation of Atg2a. As a consequence, mTOR was activated and AMPK was suppressed, resulting in the inhibition of autophagy. This dysregulation of AMPK/mTOR affected the autophagic flux, which is hallmarked by LC3B accumulation and increased SQSTM1 protein levels. Autophagy is involved in proacrosomal vesicle fusion and transport to form the acrosome. We conclude that this disruption leads to the observed malformation of the acrosome. TRIM27 is associated with PFN3 as determined by co-immunoprecipitation from testis extracts. Further, actin-related protein ARPM1 was absent in the nuclear fraction of Pfn3–/– testes and sperm. This suggests that lack of PFN3 leads to destabilization of the PFN3–ARPM1 complex, resulting in the degradation of ARPM1. Interestingly, in the Pfn3–/– testes, we detected increased protein levels of essential actin regulatory proteins, cofilin-1 (CFL1), cofilin-2 (CFL2), and actin depolymerizing factor (ADF). Taken together, our results reveal the importance for PFN3 in male fertility and implicate this protein as a candidate for male factor infertility in humans.

The mammalian placenta is both the physical interface between mother and fetus, and the source of endocrine signals that target the maternal hypothalamus, priming females for parturition, lactation and motherhood. Despite the importance of this connection, the effects of altered placental signaling on the maternal brain are understudied. Here, we show that placental dysfunction alters gene expression in the maternal brain, with the potential to affect maternal behavior. Using a cross between the house mouse and the Algerian mouse in which hybrid placental development is abnormal, we sequenced late gestation placental and maternal medial preoptic area transcriptomes and quantified differential expression and placenta-maternal brain co-expression between normal and hybrid pregnancies. The expression of Fmn1, Drd3, Caln1 and Ctsr was significantly altered in the brains of females exposed to hybrid placentas. Most strikingly, expression patterns of placenta-specific gene families and Drd3 in the brains of house mouse females carrying hybrid litters matched those of female Algerian mice, the paternal species in the cross. Our results indicate that the paternally-derived placental genome can influence the expression of maternal-fetal communication genes, including placental hormones, suggesting an effect of the offspring’s father on the mother’s brain.

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and mediates chromatin hyper-condensation, together with PRM1. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation and the prevention of DNA damage. Yet, the role of cP2 is not yet understood. Using CRISPR-Cas9 mediated gene editing, we generated mice lacking the cP2 domain while the mP2 is still expressed. We show that deletion of one allele of the cP2 domain is sufficient to render male mice infertile. cP2 deficient sperm show incomplete PRM2 incorporation, retention of transition proteins and a severely altered protamine ratio. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation, inviability and immotility of mature sperm. The cP2 domain therefore seems to be necessary for the complex crosstalk leading to the successive and complete removal of transition proteins and complete protamination of sperm chromatin. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research.

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