Differential distribution of 5-formylcytosine and 5-carboxylcytosine in human spermatogenic cells and spermatozoa

Cover Page


Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription or Fee Access

Abstract

Background. The epigenome of gametes is formed under the control of the developmental programme and the influence of environmental factors. How cytosine oxidation patterns are formed and altered in human spermatogenesis remains obscure so far.

The aim of the study was to assess 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) patterns in human spermatogenic cells and spermatozoa.

Materials and Methods. The study was performed on testicular biopsy samples of 10 azoospermic patients and ejaculate samples of 5 sperm donors and 8 patients from infertile couples. The microscope slides were prepared for further indirect immunofluorescence to detect 5fC and 5caC and FISH to determine spermatogenic cell ploidy.   

Results. 5fC and 5caC were undetectable in mitotic and meiotic chromosomes of spermatogenic cells, and was present exclusively in some spermatogonia and spermatid interphase nuclei as well as in some ejaculated spermatozoa. The frequency of spermatozoa with 5fC and 5caC varied in a wide range and was higher in patients than in sperm donors (p=0,007, p=0,028). The increase in frequency of spermatozoa with 5fC and 5caC was accompanied with the decrease in frequency of morphologically normal and progressively motile spermatozoa.

Conclusions. 5fC and 5caC are differentially distributed in human spermatogenic cells and spermatozoa. The immunocytochemically detected increase of 5fC and 5caC in individual spermatozoa is most likely induced by oxidative stress caused by effects of internal and external factors rather than developmental programme. The evaluation of 5fC and 5caC in spermatozoa can be potentially used as an additional criterion of ejaculate quality.

Full Text

Restricted Access

About the authors

Olga A. Efimova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: efimova_o82@mail.ru
ORCID iD: 0000-0003-4495-0983
SPIN-code: 6959-5014
Scopus Author ID: 14013324600

Cand. Sci. (Biol.), head of Laboratory of cytogenetics and cytogenomics of reproduction

Russian Federation, Saint Petersburg

Mikhail I. Krapivin

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: krapivin-mihail@mail.ru
ORCID iD: 0000-0002-1693-5973
SPIN-code: 4989-1932
Scopus Author ID: 56507166200

junior research associate

Russian Federation, Saint Petersburg

Sergey E. Parfenyev

Saint Petersburg State University

Email: gen21eration@gmail.com
SPIN-code: 9703-0273
Scopus Author ID: 57190742512

student

Russian Federation, Saint Petersburg

Irina D. Mekina

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: irendf@mail.ru
ORCID iD: 0000-0002-0813-5845
SPIN-code: 4682-8590
Scopus Author ID: 7006299063

Cand. Sci. (Biol.), senior research associate

Russian Federation, Saint Petersburg

Evgeniia M. Komarova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: evgmkomarova@gmail.com
ORCID iD: 0000-0002-9988-9879
SPIN-code: 1056-7821
Scopus Author ID: 57191625749

Cand. Sci. (Biol.), head of Laboratory of preimplantation development

Russian Federation, Saint Petersburg

Mariia A. Ishchuk

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductologyt"

Email: mashamazilina@gmail.com
ORCID iD: 0000-0002-4443-4287
SPIN-code: 1237-6373
Scopus Author ID: 24779589100

junior research associate

Russian Federation, Saint Petersburg

Andrei V. Tikhonov

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: tixonov5790@gmail.com
ORCID iD: 0000-0002-2557-6642
SPIN-code: 3170-2629
Scopus Author ID: 57191821068

Cand. Sci. (Biol.), research associate

Russian Federation, Saint Petersburg

Igor Yu. Kogan

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: ikogan@mail.ru
ORCID iD: 0000-0002-7351-6900
SPIN-code: 6572-6450
Scopus Author ID: 56895765600

MD, Dr. Sci. (Med.), Correspondent member RAS, director

Russian Federation, Saint Petersburg

Arina V. Golubeva

Saint Petersburg State University

Email: AlikovaAV1504@yandex.ru
ORCID iD: 0000-0003-1613-222X

student

Russian Federation, Saint Petersburg

Eugene V. Daev

Saint Petersburg State University

Email: e.daev@spbu.ru
ORCID iD: 0000-0003-2036-6790
SPIN-code: 8926-6034
Scopus Author ID: 6701779129

Dr. Sci. (Biol.), professor of genetics and biotechnology Department

Russian Federation, Saint Petersburg

Aleksander M. Gzgzyan

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: agzgzyan@gmail.com
SPIN-code: 6412-4801
Scopus Author ID: 56232643300

MD, Dr. Sci. (Med.)

Russian Federation, Saint Petersburg

Olesya N. Bespalova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: shiggerra@mail.ru
SPIN-code: 4732-8089
Scopus Author ID: 57189999252

Dr. Sci. (Med.); deputy director for science

Russian Federation, Saint Petersburg

Anna A. Pendina

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology; Saint Petersburg State University

Author for correspondence.
Email: pendina@mail.ru
ORCID iD: 0000-0001-9182-9188
SPIN-code: 3123-2133
Scopus Author ID: 6506976983

Cand. Sci. (Biol.), senior research associate

Russian Federation, Saint Petersburg; Saint Petersburg

References

  1. Reik W, Surani MA. Germline and Pluripotent Stem Cells. Cold Spring Harb Perspect Biol. 2015;7(11): a019422. doi: 10.1101/cshperspect.a019422
  2. Efimova OA, Pendina AA, Tikhonov AV, Baranov VS. The evolution of ideas on the biological role of 5-methylcytosine oxidative derivatives in the mammalian genome. Russ J Genet Appl Res. 2018;8(1):11–21. doi: 10.1134/S2079059718010069
  3. Efimova OA, Koltsova AS, Krapivin MI, et al. Environmental Epigenetics and Genome Flexibility: Focus on 5-Hydroxymethylcytosine. Int J Mol Sci. 2020;21(9):3223. doi: 10.3390/ijms21093223
  4. Aitken RJ, Gibb Z, Baker MA, et al. Causes and consequences of oxidative stress in spermatozoa. Reprod Fertil Dev. 2016;28(1–2): 1–10. doi: 10.1071/RD15325
  5. Gunes S, Arslan MA, Hekim GNT, Asci R. The role of epigenetics in idiopathic male infertility. J Assist Reprod Genet. 2016;33(5): 553–569. doi: 10.1007/s10815-016-0682-8
  6. Jenkins TG, Aston KI, Meyer TD, et al. Decreased fecundity and sperm DNA methylation patterns. Fertil Steril. 2016;105(1): 51–7.e1–3. doi: 10.1016/j.fertnstert.2015.09.013
  7. Aston KI, Uren PJ, Jenkins TG, et al. Aberrant sperm DNA methylation predicts male fertility status and embryo quality. Fertil Steril. 2015;104(6):1388–97.e1–5. doi: 10.1016/j.fertnstert.2015.08.019
  8. Urdinguio RG, Bayón GF, Dmitrijeva M, et al. Aberrant DNA methylation patterns of spermatozoa in men with unexplained infertility. Hum Reprod. 2015;30(5):1014–1028. doi: 10.1093/humrep/dev053
  9. Du Y, Li M, Chen J, et al. Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia. Hum Reprod. 2016;31(1):24–33. doi: 10.1093/humrep/dev283
  10. Laqqan MM, Yassin MM. Cigarette heavy smoking alters DNA methylation patterns and gene transcription levels in humans spermatozoa. Environ Sci Pollut Res Int. 2022;29(18):26835–26849. doi: 10.1007/s11356-021-17786-8
  11. Li J, Xu J, Yang T, et al. Genome-wide methylation analyses of human sperm unravel novel differentially methylated regions in asthenozoospermia. Epigenomics. 2022;14(16):951–964. doi: 10.2217/epi-2022-0122
  12. Zhang Z, Wang J, Shi F, et al. Genome-wide alternation and effect of DNA methylation in the impairments of steroidogenesis and spermatogenesis after PM2.5 exposure. Environ Int. 2022;169:107544. doi: 10.1016/j.envint.2022.107544
  13. Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324(5929):930–935. doi: 10.1126/science.1170116
  14. Ito S, Shen L, Dai Q, et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011;333(6047):1300–1303. doi: 10.1126/science.1210597
  15. Branco MR, Ficz G, Reik W. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat Rev Genet. 2011;13(1):7–13. doi: 10.1038/nrg3080
  16. Pfeifer GP, Kadam S, Jin SG. 5-hydroxymethylcytosine and its potential roles in development and cancer. Epigenetics Chromatin. 2013;6(1):10. doi: 10.1186/1756-8935-6-10
  17. Efimova OA, Pendina AA, Tikhonov AV, et al. Oxidized form of 5-methylcytosine — 5-hydroxymethylcytosine: a new insight into the biological significance in the mammalian genome. Russ J Genet Appl Res. 2015;5:75–81. doi: 10.1134/S2079059715020033
  18. Efimova OA, Pendina AA, Tikhonov AV, et al. Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality. Oncotarget. 2017;8(51):88294–88307. doi: 10.18632/oncotarget.18331
  19. Zheng H, Zhou X, Li DK, et al. Genome-wide alteration in DNA hydroxymethylation in the sperm from bisphenol A-exposed men. PLoS One. 2017;12(6): e0178535. doi: 10.1371/journal.pone.0178535
  20. Nettersheim D, Heukamp LC, Fronhoffs F, et al. Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development. PLoS One. 2013;8(12): e82881. doi: 10.1371/journal.pone.0082881
  21. Kruger TF, Menkveld R, Stander FS, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril. 1986;46(6):1118–1123. doi: 10.1016/s0015-0282(16)49891-2
  22. Pendina AA, Efimova OA, Chiryaeva OG, et al. A comparative cytogenetic study of miscarriages after IVF and natural conception in women aged under and over 35 years. J Assist Reprod Genet. 2014;31(2):149–155. doi: 10.1007/s10815-013-0148-1
  23. Efimova OA, Pendina AA, Tikhonov AV, et al. Chromosome hydroxymethylation patterns in human zygotes and cleavage-stage embryos. Reproduction. 2015;149(3):223–233. doi: 10.1530/REP-14-0343
  24. Pendina AA, Efimova OA, Tikhonov AV, et al. Immunofluorescence Staining for Cytosine Modifications Like 5-Methylcytosine and Its Oxidative Derivatives and FISH. In: Fluorescence in situ hybridization (FISH). Ed. by T. Liehr. Berlin: Heidelberg: Springer; 2017. P. 337–346. doi: 10.1007/978-3-662-52959-1_35
  25. Efimova OA, Pendina AA, Krapivin MI, et al. Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells. Cytogenet Genome Res. 2018;156(3):150–157. doi: 10.1159/000493906
  26. Negoescu A, Lorimier P, Labat-Moleur F, et al. In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations. J Histochem Cytochem. 1996;44(9):959–968. doi: 10.1177/44.9.8773561
  27. World Health Organization. Laboratory manual for the examination and processing of human semen, sixth edition. Geneva: World Health Organization; 2021. 276 p.
  28. Seisenberger S, Peat JR, Hore TA, et al. Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Philos Trans R Soc Lond B Biol Sci. 2013;368(1609):20110330. doi: 10.1098/rstb.2011.0330
  29. Marcho C, Cui W, Mager J. Epigenetic dynamics during preimplantation development. Reproduction. 2015;150(3):R109–R120. doi: 10.1530/REP-15-0180
  30. Fulka H, Mrazek M, Tepla O, Fulka J Jr. DNA methylation pattern in human zygotes and developing embryos. Reproduction. 2004;128(6):703–708. doi: 10.1530/rep.1.00217
  31. Pendina AA, Efimova OA, Fedorova ID, et al. DNA methylation patterns of metaphase chromosomes in human preimplantation embryos. Cytogenet Genome Res. 2011;132(1–2):1–7. doi: 10.1159/000318673
  32. Madugundu GS, Cadet J, Wagner JR. Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA. Nucleic Acids Res. 2014;42(11):7450–7460. doi: 10.1093/nar/gku334
  33. Cadet J, Wagner JR. Radiation-induced damage to cellular DNA: Chemical nature and mechanisms of lesion formation. Radiation Physics and Chemistry. 2016;128:54–59. doi: 10.1016/j.radphyschem.2016.04.018
  34. Alahmar AT. Role of Oxidative Stress in Male Infertility: An Updated Review. J Hum Reprod Sci. 2019;12(1):4–18. doi: 10.4103/jhrs.JHRS_150_18
  35. Ni K, Dansranjavin T, Rogenhofer N, et al. TET enzymes are successively expressed during human spermatogenesis and their expression level is pivotal for male fertility. Hum Reprod. 2016;31(7):1411–1424. doi: 10.1093/humrep/dew096
  36. Sakkas D, Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril. 2010;93(4):1027–1036. doi: 10.1016/j.fertnstert.2009.10.046

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Interphase nuclei of spermatogenic cells from azoospermic patient after hybridization with DNA probe specific to centromeric region of chromosome 8 (aqua) and subsequent immunodetection of 5fC (yelow) and 5caC (green). DNA is stained with DAPI (blue). Four areas on the preparation are shown (a–d)

Download (258KB)
Indexing metadata
3. Fig. 2. The ratio of interphase nuclei with 5fC and 5caC among spermatogonia and spermatids in 10 azoospermic patients

Download (250KB)
Indexing metadata
4. Fig. 3. Interphase nucleus of spermatogenic cell from testicular biopsy of azoospermic patient: a — immunocytochemical detection of 5fC*; b — immunocytochemical detection of 5caC; c — merged image (5fC + 5caC). *To improve quality of the merged image, picture a was converted from orange to red pseudocolor

Download (164KB)
Indexing metadata
5. Fig. 4. Human ejaculated spermatozoa stained with anti-5fC (a)* and anti-5caC (b) antibodies, merged image — 5fC + 5caC (c), phase contrast image (d). *To improve quality of the merged image, picture a was converted from orange to red pseudocolor

Download (139KB)
Indexing metadata
6. Fig. 5. Positive correlation between the frequency of 5fC+ and 5caC+-spermatozoa in ejaculate (Spearman test)

Download (48KB)
Indexing metadata
7. Fig. 6. Scatter dot plots showing the frequency of immunocytochemically detected 5fC+ and 5caC+-spermatozoa in ejaculate of patients from infertile couples and sperm donors

Download (96KB)
Indexing metadata
8. Рис. 7. Сперматозоид из эякулята человека, окрашенный с помощью антител, специфичных к 5fC (a)*, 5caC (b), и совмещенные изображения 5fC + 5caC (c). *Для улучшения качества совместной визуализации флуоресцентных сигналов изначально оранжевый цвет на изображении a был изменен в фоторедакторе на красный

Download (96KB)
Indexing metadata
9. Fig. 8. Correlations between the frequency of 5fC+ / 5caC+-spermatozoa in ejaculate and semen parameters. Statistically significant correlations are framed (Spearman test)

Download (161KB)
Indexing metadata
10. Fig. 9. Correlations between the frequency of 5fC+ / 5caC+-spermatozoa in ejaculate and DNA fragmentation (Spearman test)

Download (70KB)
Indexing metadata

Copyright (c) 2023 Eco-Vector


СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 65617 от 04.05.2016.


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies