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.

P–115 Supplementation of healthy Sertoli cells into culture media containing follicle stimulating hormone (FSH)/testosterone (T) has no advantage in germ cell maturation

Study question In nonobstructive azoospermia (NOA) cases, whether supplementation of healthy Sertoli cells (SCs) has an effect on spermatogenic differentiation in culture medium containing FSH/T. Summary answer Expression of Crem and Acrosin increased significantly in both medium with FSH/T and medium with additional healthy SCs but there was no difference between them What is known already In NOA the induction of spermatogonial stem cells (SSCs) proliferation and differentiation has been demonstrated using different culture systems. SCs have vital roles in the regulation of spermatogenesis. Hormonal control of spermatogenesis is through FSH and T activity on SCs. Growth factors secreted by SCs via FSH, stimulate proliferation and colonization of SSCs. Although germ cells do not express androgen receptors, FSH receptors are localized on spermatogonia. It is not clear whether native SCs are sufficient for FSH/T added to the culture medium to be effective in induction of spermatogenesis, and whether supplementation of healthy SCs will increase this activity. Study design, size, duration 34 NOA and 12 obstructive azoospermia (OA) cases were included. Testicular tissue samples were taken with testicular sperm extraction (TESE) in the study and control groups. In a group of fertile cases, healthy Sertoli cells were identified and purified and then cryopreserved. Tissue samples of each case prepared in standard DMEM/F12 medium were processed in 2 separate environments containing FSH/T and FSH/T plus thawed healthy SCs for 7 days. Participants/materials, setting, methods The characterization of healthy SCs isolated from fertile cases was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. FC was used to measure the 7th day levels of specific markers expressed in spermatogonia (Vasa), meiotic cells (Crem) and post-meiotic cells (Protamine–2 and Acrosin). Main results and the role of chance In Annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT exhibited that cryopreservation didn’t inhibite the SCs proliferation compared to the pre-freezing state. Vasa and Acrosin basal levels were found to be lower in infertile patients compared to the control group (8.2% vs. 30.6% and 12.8% vs. 30.5%, p &lt; 0.05). Compared to day 0 measurements, on the 7th day in FSH/T environment, Crem level increased by 58.8% and Acrosin level increased by 195.5% (p &lt; 0.05). Similarly, in medium supplemented with healthy SCs, by day 7, the Crem and Acrosin levels were increased to 92.2% and 204.8%, respectively (p &lt; 0.05). Although Vasa and Protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the 7th day increase rates of Crem, Vasa, Acrosin and Protamine–2 in either FSH/T-containing medium or in medium additionally supplemented with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8% and 232.3% vs. 198.4%, respectively, p &gt; 0.05). Our results suggest that freezing-thawing process would not impair the viability and proliferation of SCs, and adding healthy SCs to the culture medium to correct impaired gene expression does not have an advantage over FSH/T. Limitations, reasons for caution The 7-day culture period we determined might be not sufficient for spermatogenic differentiation completion. This period could be extended in order to see further morphological differentiation may need. Wider implications of the findings: The failure of the culture media containing FSH/T to show the expected effectiveness could be thought to be due to the SCs’ inadequate response to these hormones. Therefore, healthy SCs supplementation would be needed, but this could pose ethical issues. Our findings show that it is not necessary. Trial registration number 214S532

Protamine-2 Deficiency Initiates a Reactive Oxygen Species (ROS)-Mediated Destruction Cascade during Epididymal Sperm Maturation in Mice

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.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l−/−; Prm2S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l−/− mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.