Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities Csilla Krausz 1,2 * . , Juan Sandoval 3. , Sergi Sayols 3 , Chiara Chianese 1 , Claudia Giachini 1 , Holger Heyn 3 , Manel Esteller 3,4,5 * 1 Department of Clinical Physiopathology, Andrology Unit, University of Florence, Florence, Italy, 2 Fundacio Puigvert, Barcelona, Catalonia, Spain, 3 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain, 4 Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain, 5 Institucio Catalana de Recerca i Estudis Avanc ¸ats (ICREA), Barcelona, Catalonia, Spain Abstract Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter- individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research. Citation: Krausz C, Sandoval J, Sayols S, Chianese C, Giachini C, et al. (2012) Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities. PLoS ONE 7(10): e44479. doi:10.1371/journal.pone.0044479 Editor: Joel R. Drevet, Clermont-Ferrand Univ., France Received May 22, 2012; Accepted August 7, 2012; Published October 2, 2012 Copyright: ß 2012 Krausz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the European Research Council (ERC) Advanced Grant EPINORC, the Ministerio de Ciencia e Innovacio ´ n (MICINN) grant SAF2011-22803, the Health Department of the Catalan Government (Generalitat de Catalunya), the Italian Ministry of University (grant PRIN 2010-2012 to CK), the Spanish Ministry of Health (grant FIS -PI11/02254). JS is a ‘‘Juan de la Cierva’’ Researcher. ME is an Institucio Catalana de Recerca i Estudis Avanc ¸ats (ICREA) Research Professor. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: c.krausz@dfc.unifi.it (CK); mesteller@idibell.cat (ME) . These authors contributed equally to this work. Introduction Human spermatogenesis is an outstandingly complex biological process which requires the concerted action of several thousands of genes [1]. An interesting feature of this biological process is the extremely large inter-individual variability of sperm production in healthy fertile men. The entity of this variation is well illustrated by a large recent study, reporting that total sperm number in the so called normal range (defined as 5 th -95 th percentile), varies from 40 millions to several hundred millions [2]. While a few genetic variants have been studied in relation to spermatogenic efficiency in normozoospermic men [3–6], the epigenetic aspects of such variations in the normozoospermic range is completely unex- plored. Apart from the large inter-individual variability of the above mentioned quantitative traits of spermatogenesis, semen of normozoospermic men contains a qualitatively (in terms of motility and morphology) heterogeneous sperm population. With the advent and diffusion of assisted reproductive techniques, a number of sperm selection methods have been developed in order to obtain sperm subpopulations enriched with highly motile and morphologically normal spermatozoa to be used for in vivo or in vitro insemination. The rationale behind selection is mainly related to a predicted higher functional competency and a higher genomic integrity of selected spermatozoa. Interestingly enough, despite the same testicular environment, biochemical markers [7,8] as well as DNA integrity [9–12] show differences in distinct sperm fractions belonging to the same individual. It is still unknown whether these fractions also show differences in their methylation level. Given that epigenetic signals such as DNA methylation and histone modifications are crucial for the proper functioning of the genome, phenotypic differences in sperm production (quantitative as well as qualitative traits) at both inter- and intra-individual level may also be due to an epigenetic variation. This hypothesis seems PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e44479