Single residue substitution in protamine 1 disrupts sperm genome packaging and embryonic development in mice

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, an alanine substitution (P1 K49A) results in ectopic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin. In vitro, the P1 K49A substitution decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential to ensure reproductive fitness.

Potential effect of tobacco cigarettes smoking on global DNA methylation status and protamines transcripts in human spermatozoa

Background Epigenetics refers to an alteration in gene expression without alteration in the sequence of DNA and this process may be affected by environmental factors and lifestyle like cigarette smoking. This study was designed to evaluate the potential effect of cigarette smoking on the global DNA methylation status and the transcription level of protamine 1 and protamine 2 in human spermatozoa. A total of 188 semen samples were collected from men with a mean age of 34.9 ± 5.8 years old (98 heavy smokers and 90 non-smokers). The DNA and RNA were isolated from purified spermatozoa, then the status of global DNA methylation and the transcription level of protamine 1 and protamine 2 were evaluated using ELISA and qPCR, respectively. The chromatin non-condensation and DNA fragmentation in human spermatozoa were evaluated using chromomycin A3 staining and TUNEL assay, respectively. Results A significant increase has been found in the status of global DNA methylation in spermatozoa of heavy smokers compared to non-smokers (7.69 ± 0.69 ng/μl vs. 4.90 ± 0.40 ng/μl, P < 0.001). Additionally, a significant reduction has been found in transcription level of protamine 1 (25.49 ± 0.31 vs. 23.94 ± 0.40, P < 0.001) and protamine 2 (28.27 ± 0.39 vs. 23.45 ± 0.30, P < 0.001) in heavy smokers. A downregulation has been found in the transcription level of protamine 1 and protamine 2 with a fold change of 0.497 and 0.047, respectively. A significant increase has been shown in the level of DNA fragmentation and chromatin non-condensation in heavy smokers compared to non-smokers (P < 0.001). On the other hand, a significant positive correlation has been found between sperm chromatin non-condensation, sperm DNA fragmentation, transcription level of protamine 1, transcription level of protamine 2, and global DNA methylation status (r = 0.304, P < 0.001; r = 0.399, P < 0.001; r = 0.216, P = 0.003; r = 0.494, P < 0.001, respectively). Conclusion Tobacco cigarette smoking has a potential influence on the global DNA methylation and the transcription level of protamine genes in human spermatozoa, and consequently, affect negatively on the semen parameters.

P–051 Differential resilience of sperm from different mammals to DNA decondensation

Study question Does sperm from different species with different protamine 1/protamine 2 ratios have different resilience to sperm decondensation? Summary answer Sperm cells from species whose DNA is condensed with both protamine 1 and protamine 2 require less time in deprotamination steps. What is known already Sperm cells present a highly particular DNA condensation that is acquired during sperm differentiation, where most part of histones are replaced by protamines. Protamines are key elements for DNA condensation and, while protamine 1 is more conserved among species, protamine 2 has evolved differentially, existing only a few species that retain the mature protein in their sperm DNA. Changes in protamine expression rates have been described to be associated to head sperm size and shape. In addition, reduced amounts of protamine 2 are related to male infertility in species in which this protein is present. Study design, size, duration Cryopreserved sperm samples were treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. In these treatments, the effect of different incubation times with proteinase K added to the lysis solution upon DNA decondensation was tested by analyzing core diameter, halo diameter and the Halo/core ratio in at least 50 sperm per sample. Participants/materials, setting, methods Species included in the study were Human, Equine, Donkey, Porcine and Bovine. Sperm samples from five different individuals for each species were included in the study. DNA decondensation included three lysis steps: first, a SDS + DTT incubation for 30 minutes; second, a DTT + NaCl treatment for 30 minutes; and third, a DTT + NaCl + Proteinase K treatment with a variable time of 0, 30 or 180 minutes. Main results and the role of chance The halo/core diameter, used as a representation of the degree of DNA decondensation, for 0 minutes, 30 minutes and 180 minutes of proteinase K incubation were: 4.68±0.51, 4.32±0.51 and 4.77±0.64, respectively for human sperm; 4.15±0.41, 4.57±0.53 and 4.68±0.63, respectively for Equine sperm; 4.40±0.64, 4.00±0.37 and 4.17±0.19, respectively for donkey sperm; 1.77±0.2, 3.05±0.14 and 4.13±0.39, respectively for porcine sperm; and 2.40±0.40, 3.36±0.22 and 4.19±0.38, respectively for bovine sperm. Differences of halo/core ratio in different times were only observed in porcine and bovine sperm, where increasing degrees of DNA decondensation were found (p &lt; 0.05). Therefore, these results show that while longer incubations in lysis solutions with proteinase K lead to higher DNA decondensation in porcine and bovine, they do not induce higher decondensation in human, equine and donkey. This evidence, coupled to the fact that porcine and bovine sperm present null or very low protamine 2 content, suggests that its presence might confer higher DNA decondensation susceptibility. Limitations, reasons for caution Only sperm cells with normal sperm haloes were analyzed in the present study. As multiple studies show, haloes exhibited by sperm cells with DNA damage display higher diameter, that is why they were strictly excluded in this study with the aim to elucidate the average DNA decondensation. Wider implications of the findings: Sperm DNA might have different degrees of DNA condensation, which can be associated to a higher difficulty of DNA decondensation, thus having implications in the sensitivity tests that assess sperm DNA integrity. Trial registration number Not applicable.

The study of sperm head vacuoles using deep learning algorithm and its correlation with protamine mRNA ratio

BackgroundAs regards the routine semen analysis is not sufficient to assess male fertility status, is it necessary to use other morphological sperm examination that may be more relevant in regard to the promotion of assisted reproduction outcomes? This study was designed for examination of sperm vacuole characteristics, its association with other sperm parameters and protamine 1 to protamine 2 ratio, and predict assisted pregnancy outcomes. Methods 98 Semen samples from subfertile men were classified based on Vanderzwalmen's criteria as follows: grade I, no vacuoles; grade II, ≤ 2small vacuoles; grade III, ≥ 1 large vacuole; grade IV, large vacuole with other abnormalities. The location, frequency and size of vacuoles were assessed using high magnification, a deep learning algorithm, and scanning electron microscope methods. The chromatin integrity (toluidine blue staining), condensation status (aniline blue), viability and acrosome integrity (triple staining), and protamination status (CMA3 staining) was evaluated for vacuolated samples. Protamine 1 and protamine 2 gene expression was analyzed by quantitative real-time PCR. The assisted reproduction outcomes were also followed for each cycle. Results The results show a significant correlation between the vacuole size (III and IV) and abnormal sperm chromatin condensation (p<0.05), and protamine-deficient (p<0.05). The percentage of reacted acrosomes was significantly higher in spermatozoa with grades III and IV compared with normal group (p<0.05). A high protamine mRNA ratio ( prm-2 was underexpressed) was observed in the vacuolated spermatozoa with grade IV (p<0.01). The sperm head vacuole was negatively associated with the fertilization rate (p<0.01) under IVF cycles. This association was also significantly observed in pregnancy and live birth rate in the groups with grade III and IV (P<0.05). Conclusions The results of our study highlight the importance of follow up of more sperm parameters such as sperm head vacuole characteristics, because may reflect protamine-deficient and poor IVF/ICSI outcomes.