ORIGINAL RESEARCH published: 30 April 2021 doi: 10.3389/fcell.2021.669182 Species-Specific Differences in Sperm Chromatin Decondensation Between Eutherian Mammals Underlie Distinct Lysis Requirements Jordi Ribas-Maynou 1,2* , Estela Garcia-Bonavila 1,2 , Carlos O. Hidalgo 3 , Jaime Catalán 4 , Jordi Miró 4 and Marc Yeste 1,2 1 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, Girona, Spain, 2 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, Girona, Edited by: Spain, 3 Department of Animal Selection and Reproduction, Regional Agrifood Research and Development Service Silvina Perez-Martinez, of Asturias (SERIDA), Gijón, Spain, 4 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty CONICET Centro de Estudios of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain Farmacológicos y Botánicos (CEFYBO), Argentina Sperm present a highly particular DNA condensation that is acquired during their Reviewed by: differentiation. Protamines are key elements for DNA condensation. However, whereas Juan Carlos Calvo, CONICET Instituto de Biología y the presence of protamine 1 (P1) is conserved across mammalian species, that Medicina Experimental (IBYME), of protamine 2 (P2) has evolved differentially, existing only few species that use Argentina Xin Zhiguo Li, both protamines for sperm DNA condensation. In addition, altered P1/P2 ratios and University of Rochester, United States alterations in the expression of P1 have previously been associated to infertility and DNA *Correspondence: damage disorders. On the other hand, different methods evaluating DNA integrity, such Jordi Ribas-Maynou as Sperm Chromatin Dispersion (SCD) and Comet tests, need a previous complete DNA jordi.ribasmaynou@udg.edu orcid.org/0000-0002-9101-2044 decondensation to properly assess DNA breaks. Related with this, the present study aims to analyze the resilience of sperm DNA to decodensation in different eutherian Specialty section: This article was submitted to mammals. Sperm samples from humans, horses, cattle, pigs and donkeys were used. Cell Growth and Division, Samples were embedded in low melting point agarose and treated with lysis solutions to a section of the journal induce DNA decondensation and formation of sperm haloes. The treatment consisted Frontiers in Cell and Developmental Biology of three steps: (1) incubation in SDS + DTT for 30 min; (2) incubation in DTT + NaCl Received: 18 February 2021 for 30 min; and (3) incubation in DTT + NaCl with or without proteinase K for a variable Accepted: 12 April 2021 time of 0, 30, or 180 min. How incubation with the third lysis solution (with or without Published: 30 April 2021 proteinase K) for 0, 30, and 180 min affected DNA decondensation was tested through Citation: Ribas-Maynou J, analyzing core and halo diameters in 50 sperm per sample. Halo/core length ratio was Garcia-Bonavila E, Hidalgo CO, used as an indicator of complete chromatin decondensation. While incubation time with Catalán J, Miró J and Yeste M (2021) the third lysis solution had no impact on halo/core length ratios in species having P1 Species-Specific Differences in Sperm Chromatin Decondensation and P2 (human, equine and donkey), DNA decondensation of pig and cattle sperm, Between Eutherian Mammals which only present P1, significantly (P < 0.05) increased following incubation with the Underlie Distinct Lysis Requirements. Front. Cell Dev. Biol. 9:669182. third lysis solution for 180 min. In addition, the inclusion of proteinase K was found doi: 10.3389/fcell.2021.669182 to accelerate DNA decondensation. In conclusion, longer incubations in lysis solution Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species including proteinase K lead to higher DNA decondensation in porcine and bovine sperm. This suggests that tests intended to analyze DNA damage, such as halo or Comet assays, require complete chromatin deprotamination to achieve high sensitivity in the detection of DNA breaks. Keywords: sperm, DNA condensation, DNA damage, protamine, mammals INTRODUCTION Protamines are arginine-rich proteins sized about half of the size of a typical histone and with highly positive charge, Different proteins encoded in nuclear and mitochondrial DNA features that result in a tight binding to the nuclear sperm allow cells to accomplish multiple functions to survive and DNA, neutralizing the phosphodiester backbone (Balhorn, 1982). proliferate. Nuclear chromatin consists of a complex of DNA and Protamine-DNA complex is responsible for the higher level of histone octamers. Histone octamers are formed by histones 2A, DNA condensation of spermatozoa compared to somatic cells; 2B, 3, and 4 (H2A, H2B, H3, and H4) and are separated with nuclear sperm DNA is known to be about six times more linker DNA of about 20–60 base pairs; each octamer condenses condensed than mitotic chromosomes, resulting in a nucleus around 146 base pairs of DNA. The DNA-histone complex is with about 44 times less volume than that of a mouse liver highly supercoiled, forms chromatin fibers of 100–400 Kb in cell (Ward and Coffey, 1991). Due to this DNA condensation, length and is organized in loop domains (Luger et al., 1997; sperm chromatin is transcriptionally silent and, after fertilization, Rao et al., 2014; Gibson et al., 2019). According to the models the replacement of protamines by histones is one of the of chromatin organization, in each of these loops can one find main processes happening in the male pronuclear stage (Ajduk a matrix attachment region (MAR) whose function is attaching et al., 2006; Ward, 2010). In mammals, two different types of chromatin to the nuclear matrix, a protein scaffold that supports protamines (protamine 1 and protamine 2 family) are encoded nuclear organization (Ward, 2010) and contributes to the in the genome. While protamine 1 is present in sperm from regulation of DNA replication and transcription in somatic cells, all species, protamine 2 family, formed by P2, P3, and P4 providing epigenetic plasticity for gene expression (Getzenberg, components, is only found in species such as humans, mice and 1994; Linnemann et al., 2009; Ji et al., 2013; Narwade et al., 2019). horses (Fuentes-Mascorro et al., 2000; Corzett et al., 2002; Oliva, Spermatozoa are highly differentiated cells whose main 2006; Gosálvez et al., 2011). Whereas protamine 1 is synthesized function is the transport of genetic material to the oocyte through as a mature protein, those belonging to protamine 2 family the female reproductive tract. To achieve this, millions of years (mainly P2, P3, and P4 are present in a minor percentage) need of evolution have conferred mammalian sperm with highly post-translational proteolysis prior to conversion into the mature specialized characteristics, such as the presence of flagellum, form (Oliva, 2006). As Protamine 2 family proteins have never organization of mid-piece, and a very particular chromatin been found to compact sperm DNA alone, they appear not to condensation and organization, as DNA is mainly condensed be capable of condensing sperm chromatin without protamine 1 in protamines rather than in histones (Ward and Coffey, 1991; (Corzett et al., 2002). Alterations in the expression of protamine Wykes and Krawetz, 2003; Oliva, 2006). Organization of sperm 1 have been shown to be linked to sperm morphology anomalies chromatin changes during spermatogenesis, as histones are and premature DNA condensation during sperm differentiation replaced by protamines through a series of events that, in some (Peschon et al., 1989; Lee et al., 1995); alterations in protamine species such as humans, mouse, rat or sheep, involve transition 2 content lead to male infertility and DNA damage (Aoki et al., nuclear proteins (Fuentes-Mascorro et al., 2000; Kierszenbaum, 2005; Nanassy et al., 2011), as confirmed by Prm2± knockouts 2001; Hao et al., 2019). Condensation of sperm DNA through (Oliva and Dixon, 1991; Cho et al., 2001). protamines protects genetic information from damaging agents The currently accepted model for chromatin organization (Zini and Libman, 2006), facilitating the delivery of an intact in mammalian sperm purports that protamine-DNA complex genetic material to the future embryo. This is very relevant forms toroidal structures, which condense about 48 kb of DNA, as sperm DNA damage has been proposed as an important and are estimated to measure between 60 and 100 nm in affectation that may underlie fertilization failure, poor embryo diameter and 20 nm in thickness (Brewer et al., 1999, 2003; development, miscarriage and the production of unhealthy Miller et al., 2010). These toroidal structures are stabilized by offspring (Makker et al., 2009; Lewis and Simon, 2010). These disulfide covalent bonds between neighbor protamines, and are effects, however, are subject to the DNA damage repair capacity linked through uncoiled linker DNA in toroid linker regions provided by the oocyte and, in this regard, different works (TLR). The function of TLR is to attach the chromatin to the described the activation of embryo checkpoints upon fertilization nuclear matrix and is thus supposed to contain matrix attachment with damaged sperm (Adiga et al., 2007; Toyoshima, 2009). Also, regions (MAR; Ward, 2010). Toroid linker regions are assumed a recent study in humans showed that ICSI cycles performed with to be condensed in histones containing complex epigenetic sperm containing DNA damage depict lower embryo quality, information that may play a crucial role in the very early zygote pregnancy and implantation rates and higher miscarriage rates in by influencing gene expression after fertilization and during early older than in younger women (<36 years old) (Setti et al., 2021). embryo development (Jung et al., 2017; Yoshida et al., 2018). In Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species the context of sperm chromatin, localization of histone regions some protocols include incubations with proteinase K overnight in gene-rich or gene-poor DNA is a subject of much debate (Simon et al., 2014; Ribas-Maynou and Benet, 2019). Even the among scientists, as different methodologies have been shown TUNEL assay has been proposed to display higher sensitivity to lead to opposite results (Yamaguchi et al., 2018). Moreover, it following mild decondensation with DTT (Mitchell et al., 2011). has been demonstrated that different species may have different Decondensation of sperm chromatin is therefore applied to percentages of histone-condensed DNA, ranging between 3 and multiple species, with several studies linking DNA breaks to 15% in humans, between 1 and 15% in mouse or even at fertility disorders (Pérez-Llano et al., 2010; Gosálvez et al., 2011; higher rates (like 50%) in some marsupials (Fuentes-Mascorro Esteves et al., 2020; Ribas-Maynou et al., 2020). et al., 2000; Hammoud et al., 2009; Miller et al., 2010; Samans Against this background, this study aimed at comparing et al., 2014). Despite the fact that the toroidal DNA and the the decondensation capacity of sperm DNA between different linker DNA coupled to the nuclear matrix constitute the main species, some described to contain both protamine 1 and unit of chromatin structure, evidences obtained through freeze- protamine 2, and others known to have protamine 1 only. fracture, circular dichroism and atomic force microscopy support the presence of a higher superstructure of sperm chromatin, composed by parallel stacks of lamellar sheets oriented to the MATERIALS AND METHODS nuclear long axis in bulls, bucks and men (Koehler et al., 1983; Fuentes-Mascorro et al., 2000). Also, other evidences support Reagents a nodular chromatin sub-organization, sized between 100 and Unless stated, all reagents used for this study were purchased 300 nm, in mouse, human, cattle and rat sperm (Allen et al., from Sigma-Aldrich (St. Louis, MO, United States). 1993; Haaf and Ward, 1995). However, the presence of smaller nucleosome-like particles is not discarded, especially in the Sperm Sample Obtaining and nuclear periphery (Allen et al., 1996). Regardless of chromatin Cryopreservation superorganization, different studies also reported a chromosomal Human Sperm region-specific organization, since centromeres were found to Five human semen samples were obtained from healthy be positioned in the most internal parts of the sperm nucleus, volunteers that were recruited by University of Girona, Spain. whereas telomeres that remain condensed in histones appeared All donors signed an informed consent before providing their to attach to the internal nuclear membrane, positioned in the biological samples, and the study was approved by the Ethics external parts of the nucleus (Zalensky et al., 1997; Mudrak et al., Committee, Josep Trueta Hospital, Spain (Reference PTI- 2005; Zalensky and Zalenskaya, 2007). Moreover, other authors HUMA 10012018) thus complying with the Helsinki declaration showed that chromosomal territories exist in the sperm nucleus, for the research with human specimens. All men provided leading to a precise organization of chromosomal DNA (Luetjens a semen sample through ejaculation in a sterile cup. This et al., 1999; Zalenskaya and Zalensky, 2004; Foster et al., 2005). ejaculation was maintained at 37◦ C for 30 min and was While the high degree of sperm DNA condensation requires subsequently cryopreserved. protamines to be replaced by histones upon fertilization, it also Cryopreservation was performed using Test Yolk Buffer (TYB, represents a challenge for the in vitro study of sperm chromatin. 14% glycerol, 30% egg yolk, 1.98% glucose, 1.72% sodium citrate, In this sense, previous studies tested the in vivo sperm DNA and 2% glycine, pH 7.4). Briefly, each semen sample was gently decondensation of different animal species after microinjection mixed 1:1 (v:v) with TYB, poured into a 0.5-mL cryotube, and of hamster oocytes; whereas nuclei of human, mouse, chinchilla cooled down in an isopropyl alcohol bath placed within a −80◦ C and hamster sperm were properly decondensed, this was not freezer for 12 h. Afterward, cryotubes were transferred to liquid the case of cattle or rat sperm (Perreault et al., 1988). The nitrogen. Samples were thawed by submerging cryotubes in a same study showed that, while DTT and SDS led to sperm water bath at 37◦ C, and then diluted with PBS (1:3 dilution), DNA decondensation, incubation with these two agents needed added drop-by-drop in order to avoid osmotic shock. to be 10 times longer in cattle than in human sperm to achieve similar levels of decondensation (Perreault et al., 1988). Pig Sperm Nowadays, NaCl is known to be strictly necessary to remove Researchers did not manipulate animals, but semen samples were protamines from DNA, a required step to achieve complete rather purchased from a local farm (Servicios Genéticos Porcinos, DNA decondensation and form sperm haloes of decondensed S.L.; Roda de Ter, Spain), which provided commercial, standard chromatin, leaving TLR attached to the nuclear matrix (Mohar doses (90 mL) intended to artificial insemination (AI). A total et al., 2002; Sotolongo et al., 2003). of five semen samples were obtained from five sexually mature Different methods require complete sperm DNA Piétrain boars of proven fertility. Ejaculates were collected using decondensation to evaluate DNA breaks. DNA fragmentation the gloved-hand method, and the sperm rich fraction was diluted assays, such as the SCD test, rely on the differential chromatin to 3.3 × 107 spermatozoa/mL with a long-term commercial decondensation of spermatozoa containing DNA breaks extender (Vitasem LD; Magapor SL, Zaragoza, Spain). compared to those with non-damaged DNA (Fernández et al., Cryopreservation was performed using the following protocol. 2003; Cortés-Gutiérrez et al., 2009). Likewise, the Comet assay First, 50-mL aliquots were centrifuged at 2,400 × g and 15◦ C requires complete DNA decondensation for migration of for 3 min, and sperm suspensions were diluted in β-lactose- fragments and the subsequent formation of the Comet tail, and egg yolk (LEY) medium (80% v:v lactose, 20% egg yolk) to Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species a final concentration of 1.5 × 109 sperm/mL. Thereafter, all were evaluated, and samples were diluted again to reach a final aliquots were cooled down to a final temperature of 5◦ C, at concentration of 2 × 106 viable sperm/mL. Following this, a rate of −0.1◦ C/min. Then, samples were diluted to a final sperm were packaged into 0.5-mL straws and frozen with a concentration of 1 × 109 sperm/mL in LEYGO medium, which programmable freezer (Ice-Cube, Minitüb) with the following consisted of LEY medium supplemented with 6% v:v glycerol cooling rates: (i) from 20 to 5◦ C at a rate of −0.25◦ C/min; (ii) and 1.5% Orvus ES Paste (Equex STM; Nova Chemical Sales from 5 to −90◦ C at a rate of −4.75◦ C/min; and (iii) from −90 to Inc., Scituate, MA, United States). Finally, samples were packed −120◦ C at a rate of −11.11◦ C/min. Finally, straws were plunged into 0.5-mL plastic straws (Minitüb, Tiefenbach, Germany), and into liquid nitrogen and stored. a programmable controlled-rate freezer (Ice-Cube, Minitüb) was For thawing, straws were submerged in a water bath at 38◦ C used to cryopreserve samples at the following cooling rates: for 20 s, and the content was diluted with three volumes of in 6◦ C/min from 5 to –5◦ C (100 s); –39.82◦ C/min from –5 to 1.5 mL of Kenney medium at 37◦ C per straw. –80◦ C (113 s); holt at –80◦ C for 30 s; and cooled at –60◦ C/min from –80 to –150◦ C (70 s). Samples were finally plunged into Cattle Sperm liquid nitrogen. Five separate samples from five healthy and sexually mature Samples were thawed through submerging a 0.5-mL straw in Holstein bulls were used. All animals were kept in an a 38 ◦ C water bath for 15 s, with shaking. The entire volume of artificial insemination center (Cenero-Asturias, Gijón, Spain), the straw was diluted in three volumes (1:3, v:v) of pre-warmed and ejaculates were collected through an artificial vagina. Beltsville Thawing Solution (BTS). Collected semen was diluted in Bioxcell extender reaching a concentration of 92 × 106 sperm/mL. Then, samples were Horse Sperm cooled down to 4◦ C at a rate of −0.2◦ C/min, and after 3 h of Five cryopreserved straws from five separate animals were equilibration, extended semen was packaged into 0.25-mL straws used. All stallions were donors of proven fertility, and were containing 23 × 106 sperm/straw. housed at the Equine Reproduction Service, Autonomous Cryopreservation was performed in a programmable freezer University of Barcelona (Spain), which is a European Union (Digit-cool; IMV Technologies, L’Aigle, France) following the approved equine semen collection center (Authorization code: standard curve for bovine sperm: −5◦ C/min from 4 to −10◦ C, ES09RS01E). Semen samples were obtained using a Hannover- −40◦ C/min from −10 to −100◦ C, and −20◦ C/min from −100 type artificial vagina (MiniTüb, Tiefenbach, Germany) that to −140◦ C. Thawing was performed by submerging straws in a contains a nylon mesh filter to separate the ejaculate gel fraction. water bath at 38◦ C for 40 s, with shaking. The obtained ejaculates were directly diluted (1:5, v:v) in previously warmed (37◦ C) Kenney buffer (49% glucose, w:v; 24% Halo Formation powdered milk, w:v). Halo formation was induced following the same protocol for Cryopreservation was performed according to the following all species. First, samples were diluted in phosphate-buffered protocol: first, samples were centrifuged at 600 × g and 20◦ C saline (PBS) to reach a final concentration of 1 × 106 sperm/mL. for 15 min. Then, pellets were resuspended in BotuCRYO Meanwhile, an aliquot of low melting point agarose at 1% was commercial extender (Botupharma, Botucatu, Brazil) to reach a melted at 70◦ C for 10 min and warmed to 37.5◦ C for further concentration of 2 × 106 viable sperm/mL. Samples were then 10 min. Next, the sample and low melting point agarose were packaged into 0.5-mL straws and frozen in a programmable mixed at a 1:2 (v:v) ratio, obtaining a final agarose concentration freezer (Ice-Cube) as follows: (i) −0.25◦ C/min from 20 to 5◦ C of 0.66%. A drop of 6 µL of the mixture was then dispensed (60 min); (ii) −4.75◦ C/min from 5 to −90◦ C (20 min); and onto an agarose pre-treated slide, covered with a 8-mm circular (iii) −11.11◦ C/min from −90 to −120◦ C (2.7 min). Straws were coverslip, and allowed to jellify on a metal cold plate at 4◦ C finally plunged into liquid nitrogen for storage. for 5 min. Afterward, the coverslip was gently removed and Thawing was performed by submerging straws in a water bath the slide was incubated in a first lysis solution (0.8 M Tris- at 38◦ C for 20 s. The volume of each straw was diluted in three HCl, 0.8 M DTT, 1% SDS; pH = 7.5) at room temperature for volumes of pre-warmed Kenney medium (1:3, v:v). 30 min. Following this, slides were incubated in a second lysis solution (0.4 M Tris-HCl, 0.4 M DTT, 50 mM EDTA, 2 M NaCl, Donkey Sperm 1% Tween20; pH = 7.5) at room temperature for 30 min. At Five semen samples coming from five different jackasses of this point, slides were differentially incubated with a third lysis proven fertility were used. Animals were located at the Equine solution that contained proteinase K (0.4 M Tris-HCl, 0.4 M DTT, Reproduction Service, Autonomous University of Barcelona 50 mM EDTA, 2 M NaCl, 1% Tween20, 100 µg/mL proteinase (Bellaterra, Spain). Semen samples were obtained using a K; pH = 7.5). Incubation with this third lysis solution was also Hannover artificial vagina (Minitüb) with a nylon mesh filter to performed at room temperature for 0, 30, or 180 min. To assess remove the gel fraction. Immediately after collection, semen was the effect of incubating pig and cattle sperm with proteinase K, mixed with Kenney extender (1:5, v:v) at 37◦ C. decondensation with a third lysis solution that did not contain For cryopreservation, the following protocol was used: first, proteinase K (0.4 M Tris-HCl, 0.4 M DTT, 50 mM EDTA, 2 diluted semen was centrifuged at 600 × g and 20◦ C for 15 min, M NaCl, 1% Tween20, 100 µg/mL proteinase K; pH = 7.5) was and pellets were resuspended in 2 mL of INRA-freeze freezing also performed for 0, 30, or 180 min. After all lysis steps, slides extender (INRA, Paris, France). Then, concentration and motility were washed with a neutral pH solution (0.4 M Tris-HCl, pH Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species 7.5) for 5 min, and dehydrated in ethanol series (70, 90, and Armonk, NY, United States). Graphs were drawn using 100%; 2 min/step). GraphPad Prism Software (GraphPad; San Diego, CA, United States). Once data were recorded and compiled, Assessment of DNA Decondensation normal distribution and homogeneity of variances were checked Halo and core diameters are used as DNA decondensation through Shapiro-Wilk and Levene tests. Since, even after √ √ indicators. In order to obtain both measures, samples were linear transformation [log(x), arcsine x, x], data did not fit labeled with Safeview DNA stain (NBS Biologicals, Huntingdon, with parametric assumptions, Scheirer-Ray-Hare and Mann- United Kingdom) prior to evaluation under a Zeiss Imager Whitney tests were run. A significance level of 95% for the Z1 epifluorescence microscope (Carl Zeiss AG, Oberkochen, confidence interval was established to consider statistically Germany). Captures at 1,000× magnification of at least 50 significant differences. cells per experimental condition were obtained using Axiovision 4.6 software (Carl Zeiss AG, Oberkochen, Germany). In these captures, exposure time was adapted to correctly differentiate RESULTS haloes from core sperm heads. From these pictures, diameters of the core and the halo were measured as indicated in Figure 1, Figure 2 shows the formation of sperm haloes in different species, using the Image J measurement tool. These diameters were following different incubation times with proteinase K (third obtained in duplicate, and mean values were calculated for each lysis step). The application of the third lysis step containing sperm cell. In order to normalize data, a scale bar was used to proteinase K had a differential effect on the separate species obtain the correspondence of pixels to µm at the resolution used studied. Whilst human, horse and donkey sperm did not present (1,388 × 1,040 pixels). higher decondensation when incubated with proteinase K for a longer period (third lysis step), pig and cattle sperm showed Staining of Nuclear DNA in higher DNA decondensation after incubation with proteinase K and exhibited larger sperm haloes. Non-decondensed Sperm Cells In order to measure the size of nuclear DNA, a sperm sample from each species was smeared onto a glass slide, permeabilized Lysis Solutions Used for Halo Formation with PBS containing 0.25% Triton X-100 for 2 min, and stained Induce Nuclear Core Decondensation with Safeview DNA stain. Measures of nuclear core were made Following DNA staining of sperm spreads/smears, dimensions of following the same procedure depicted in Figure 1. nuclei were found to be: 4.14 µm ± 0.41 µm (mean ± standard deviation) for human sperm, 3.62 µm ± 0.55 µm for Statistics horse sperm, 3.89 µm ± 0.52 µm for donkey sperm, Data were analyzed using the Statistics Package for the 4.43 µm ± 0.34 µm for pig sperm, and 4.80 µm ± 0.34 µm for Social Sciences software (IBM SPSS ver. 25; IBM Corp., cattle sperm. While treatment with lysis solutions that did not contain proteinase K led to chromatin decondensation of sperm core, the extent of that decondensation differed between species. In effect, while, after this step, the cores of human, horse and donkey sperm were 7.11 µm ± 0.96 µm, 5.67 µm ± 0.55 µm, and 5.83 ± 0.35, respectively, they were found to measure 4.98 µm ± 0.50 µm in pig sperm and 4.34 µm ± 0.79 µm in cattle sperm. In spite of this, this increase in the diameter of the core, which indicated DNA decondensation, was statistically significant in all cases (human, P < 0.001; horse, P < 0.001; donkey, P < 0.001; pig, P < 0.001; and cattle, P = 0.004). Moreover, nuclear core also showed an increase in decondensation associated to the additional treatment of sperm cells with a lysis solution containing proteinase K (Table 1). This increase was statistically significant in horse, donkey, porcine and bovine sperm when comparing 180 min of incubation with 0 min of incubation (P < 0.05); and in human, porcine and bovine sperm when comparing 180 min of incubation with 30 min of incubation (P < 0.05). Prolonged Lysis Incubation Contributes to Higher Efficiency in Protamine Removal FIGURE 1 | Measurement of core and halo in sperm cells with decondensed Table 1 shows the effect of incubation in the third lysis solution chromatin. containing proteinase K on halo diameter. We observed that Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species FIGURE 2 | Decondensation of sperm DNA through the formation of sperm haloes. It is worth noting that porcine and bovine sperm presented higher DNA decondensation with increasing incubation times over the third lysis step containing proteinase K. In contrast, no significant changes in human, horse and donkey sperm were observed. Scale bar: 5 µm. TABLE 1 | Effect of the third lysis solution containing proteinase K on halo and core diameter in different species. Incubation time (min) Human Horse Donkey Pig Cattle Core with (µm) 0 7.11 ± 0.96 5.67 ± 0.55 5.83 ± 0.35 4.98 ± 0.50 4.34 ± 0.79 30 6.62 ± 1.17 6.92 ± 0.58a 6.32 ± 0.64 5.28 ± 0.39 6.05 ± 0.58a 180 8.19 ± 0.98b 6.96 ± 1.60a 7.15 ± 0.79a 6.37 ± 0.61a,b 7.16 ± 1.01a,b Halo + core diameter (µm) 0 32.45 ± 0.86 22.91 ± 1.46 25.18 ± 3.67 8.62 ± 1.04 10.36 ± 3.71 30 27.74 ± 2.54a 30.28 ± 1.39a 24.55 ± 2.78 15.87 ± 1.32a 20.15 ± 3.22a 180 37.76 ± 1.46a,b 30.69 ± 2.41a 29.32 ± 2.82b 25.81 ± 1.53a,b 29.17 ± 2.50a,b Halo/Core ratio 0 4.68 ± 0.51 4.15 ± 0.47 4.40 ± 0.64 1.77 ± 0.20 2.40 ± 0.40 30 4.32 ± 0.51 4.57 ± 0.53 4.00 ± 0.37 3.05 ± 0.14a 3.36 ± 0.22a 180 4.77 ± 0.64 4.68 ± 0.63 4.17 ± 0.19 4.13 ± 0.39a,b 4.19 ± 0.38a,b a Statistical differences with respect to incubation with proteinase K for 0 min (P<0.05). b Statistical differences with respect to incubation with proteinase K for 30 min (P<0.05). exposure to proteinase K led to higher halo diameters in all increase in halo/core ratios associated to longer proteinase species. In effect, there was a significant (P < 0.05) increase k treatments (P > 0.05), these ratios augmented in porcine in halo diameters of human, horse, pig and cattle sperm, when and bovine sperm (0 min vs. 30 min; 30 min vs. 180 min; 0 and 180 min of incubation were compared. Furthermore, P < 0.05). 180-min incubations compared to their 30-min counterparts also showed a significant increase in halo diameters of human, donkey, pig and cattle sperm (P < 0.05). However, and in The Inclusion of Proteinase K in the Third spite of these significant differences, the extent of the increase Lysis Solution Accelerates DNA of halo diameter in porcine and bovine sperm was much Decondensation higher (Table 1). To determine the importance of including proteinase K in the third lysis solution, we tested DNA decondensation in pig and The Halo/Core Ratio Reflects the cattle sperm after incubation with the same lysis solution without Decondensation Status of a Sperm DNA proteinase K for 30 and 180 min. Table 2 shows core diameter, Since both sperm haloes and cores showed an increased size halo diameter and halo/core ratio after such incubations. Figure 4 associated to proteinase K lysis treatment, the relationship depicts halo/core ratio as a measure of DNA decondensation. between halo size and core size appeared to be an indicator of When proteinase K was included, halo diameter significantly sperm DNA decondensation. Figure 3 and Table 1 show the increased both in pig and cattle sperm after 30 and 180 min halo/core ratio for all species involved in this study. Interestingly, of incubation (P < 0.001). Similarly, all tested conditions also while human, horse and donkey sperm did not exhibit an augmented halo/core ratios in both species (P < 0.001). Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species FIGURE 3 | Halo/core ratio in relation to proteinase K treatment (0, 30, or 180 min). ∗∗ Statistically significant differences (P < 0.05). TABLE 2 | DNA decondensation of pig and cattle sperm after incubation in the third lysis solution with or without proteinase K. Pig Cattle Incubation time (min) − Proteinase K + Proteinase K − Proteinase K + Proteinase K Core with (µm) 0 4.85 ± 0.85 4.22 ± 0.95 30 5.30 ± 0.57 5.20 ± 0.81 6.01 ± 1.03 5.95 ± 0.91 180 4.80 ± 0.53 6.24 ± 0.91** 6.63 ± 0.68 7.02 ± 1.38* Halo + core diameter (µm) 0 8.37 ± 8.37 9.87 ± 9.87 30 10.99 ± 10.99 15.49 ± 15.49** 16.20 ± 16.20 19.54 ± 19.54** 180 14.92 ± 14.92 25.63 ± 25.63** 21.40 ± 21.40 28.83 ± 28.83** Halo/Core ratio 0 1.76 ± 0.35 2.38 ± 0.62 30 2.10 ± 0.37 3.04 ± 0.54** 2.75 ± 0.51 3.32 ± 0.50** 180 3.13 ± 0.45 4.19 ± 0.72** 3.26 ± 0.45 4.23 ± 0.74** *Statistical differences with respect to incubation without proteinase K (P<0.05). **Statistical differences with respect to incubation without proteinase K (P<0.001). DISCUSSION A high DNA condensation up to almost crystalline state is a key feature that is only present in paternal germ cells and In the present study, we observed complete decondensation of allows them to transport the male genome through the female sperm DNA when samples were exposed to a lysis solution tract to reach the oocyte. This highly condensed state helps in containing proteinase K for a long period (up to 180 min). the protection of sperm cells from genotoxic damage to which We found inter-specific differences regarding sperm chromatin they are exposed (Adiga et al., 2007; Marchetti et al., 2015). decondensation following that lysis treatment since, while Due to their transcriptional silence and the loss of most of human, horse and donkey sperm did not effectively augment their cytoplasmic proteins, sperm cells are devoid of DNA repair their DNA decondensation, pig and cattle sperm achieved a systems; however, they are continuously exposed to exogenous 2.3- and 1.75-fold increase, respectively (based on halo/core and endogenous DNA damage, caused mainly through oxidative ratios). Moreover, in an additional experiment, we could establish stress (Aitken and De Iuliis, 2007). Not only are reactive that proteinase K accelerates DNA decondensation in pig and oxygen species produced by sperm mitochondria through cattle sperm. oxidative phosphorylation, which is essential for ATP generation Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species in other studies (Enciso et al., 2006) could be due to an incomplete DNA decondensation. Therefore, when complete DNA decondensation was achieved following incubation in the third lysis solution containing proteinase K for 180 min, sperm with non-fragmented DNA showed large halos, as occurs when the halo assay is used with human sperm (Fernández et al., 2005). Remarkably, our results showed that complete sperm DNA decondensation is more easily achieved in the human than in the pig. In effect, in order for similar levels of decondensed DNA to be obtained, pig sperm were observed to need a longer lysis treatment with proteinase K. In fact, proteinase K was found to act as a DNA decondensation accelerator, probably because it degrades protamines extracted by the lysis buffer (Figure 4). Previous studies using the SCD test in pig sperm lysed for 5 min, regardless of the composition of solutions (Enciso et al., 2006; Alkmin et al., 2013; Yeste et al., 2013a,b; Batista et al., 2016), are in agreement with our results. In a similar way, using the Comet assay in pig sperm without a long lysis containing proteinase K has also been suggested to lead to sensitivity-compromised results (Pérez-Llano et al., 2010; Enciso et al., 2011). Also matching with our data, a previous study aimed at elucidating alkaline labile sites in pig sperm showed the need of incubating these cells in a lysis FIGURE 4 | Halo/core ratios for incubations (0, 30, or 180 min) of pig and solution containing SDS, DTT and proteinase K for 3.5 h at 37◦ C cattle sperm in the third lysis solution without (empty dots) or with proteinase to observe the same amount of those sites in leukocytes from K (filled dots). ∗∗ Statistically significant differences (P < 0.001). the same species (Cortés-Gutiérrez et al., 2009). Furthermore, other authors incubated pig sperm with lysis solutions containing proteinase K and found that Comet images depicted complete (Camello-Almaraz et al., 2006), but can also result from the DNA decondensation (Fraser and Strzezek, 2005). presence of immature sperm, leucocytes, and other exogenous Regarding the analysis of sperm DNA integrity in cattle, effectors, such as pollutants, heat stress, infections, radiations, or previous studies showed that sperm with non-fragmented DNA other toxicants (Agarwal et al., 2008; Ribas-Maynou et al., 2020). depicted small halos, whereas those with fragmented DNA Different methods that indirectly evaluate DNA integrity presented big halos, which was in agreement with what found are based on the differential decondensation of sperm cells in pigs (García-Macías et al., 2007; Dogan et al., 2015). Our that contain DNA breaks. In this context, in vitro DNA results also indicated that bovine sperm exhibited a small halo decondensation is required in order for external and internal when either not exposed or shortly exposed to proteinase K for chromatin to be accessible. As previous studies showed, methods 30 min. However, when the time of incubation with proteinase evaluating sperm DNA integrity differ in their sensitivity to K increased, sperm chromatin of bovine sperm completely detect the actual DNA damage (Ribas-Maynou et al., 2013; Simon decondensed, in a similar fashion to what observed in other et al., 2014). In this regard, achieving complete in vitro DNA species (Figure 3). Thus, our results confirm that proteinase decondensation is critical to increase the sensitivity of methods K acts as an accelerator of DNA decondensation, and suggest evaluating its integrity, thus allowing researchers to obtain that previous studies evaluating DNA fragmentation in bovine reliable values of DNA damage. In fact, some authors explained sperm could not have decondensed DNA enough, since the lysis the low sensitivity of techniques assessing DNA integrity through period was, regardless of the solutions used, short (10 min) the hypothesis of “the iceberg tip,” which surmises that, while (García-Macías et al., 2007; Dogan et al., 2015). The use of the some sperm detected as “normal” actually have DNA breaks, Comet assay in cattle sperm also demonstrated that longer lysis they are out of the method threshold (Gosálvez et al., 2013; treatments increased the degree of DNA decondensation (Villani Cho et al., 2017). et al., 2010). However, it remains unclear whether exposure In the present study, we found that pig and cattle sperm to lysis solutions overnight in Fraser and Strzezek (2005) present higher grade of DNA condensation, which is related to could have induced artifactual DNA breaks, thus misestimating an increased difficulty to achieve its complete decondensation the actual results. in vitro and form a sperm halo in an agarose matrix (Figure 2). It is widely known that sperm DNA in humans and horses In pigs, previous studies using the SCD test considered that is condensed around both protamine 1 and protamine 2, sperm with non-fragmented DNA lacked a halo, whereas those whereas only protamine 1 but not protamine 2 is present in with fragmented DNA showed a large halo (Enciso et al., 2006; the chromatin of pig and cattle sperm (Corzett et al., 2002). Alkmin et al., 2013; Batista et al., 2016). As shown for pig Moreover, morphometric studies indicated that pig and cattle sperm after 0 min of incubation in the third lysis solution sperm display a bigger disk-shaped nucleus, whereas human and containing proteinase K (Figure 2), the lack of halo identified horse sperm are smaller and more oval-shaped (Gu et al., 2019). Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 April 2021 | Volume 9 | Article 669182 Ribas-Maynou et al. DNA Condensation in Different Mammalian Species Our results on the resilience of sperm DNA to decondensation in DATA AVAILABILITY STATEMENT the aforementioned species suggest that while sperm containing only protamine 1 display less efficiency in condensing their The original contributions presented in the study are included DNA, they have stronger protamine-protamine junctions. In in the article/Supplementary Material, further inquiries can be fact, protamine 1 in pig sperm displays the highest number directed to the corresponding author/s. of cysteine residues (10 residues) compared to other species, such as donkey (8 residues), horse (7 residues) or human (6 residues), which would support the relevance of disulfide ETHICS STATEMENT bonds between protamines in the pig (Gosálvez et al., 2011). Furthermore, bovine protamine 1 has less cysteine residues The studies involving human participants were reviewed (7 residues) than its pig counterpart, which could explain and approved by the Josep Trueta Hospital Ethics the less degree of DNA decondensation observed at 0 min Committee (Reference PTI-HUMA 10012018). The of incubation with the third lysis solution (Gosálvez et al., patients/participants provided their written informed consent 2011). Alternatively, sperm from species containing both P1 to participate in this study. and P2 appear to exhibit higher DNA condensation, with the contraindication of having less protamine-protamine unions, as different protamines from P2 family have less cysteine residues AUTHOR CONTRIBUTIONS than P1 (Corzett et al., 2002; Oliva, 2006). Moreover, when the number cysteine residues of P1 is related to the P1/P2 JR-M conceived the study, performed the experiments, analyzed ratio, the amount of cysteine residues in sperm chromatin the data, run statistics, wrote the manuscript, and approved (cysteine residues of P1 × %P1) in each species appears the final version. EG-B performed the experiments, critically to be linked to the degree of DNA condensation (halo/core revised the document, and approved the final version. CH, ratio) after 0 min of incubation with the third lysis solution JC, and JM cryopreserved semen samples, critically revised the (Supplementary Table 1). All the aforementioned could also manuscript, and approved the final document. MY contributed explain why alterations in the relative amounts of protamine 2 to the experimental design, coordinated the work, discussed lead to higher sperm DNA decondensation, and underlie DNA results, critically revised the manuscript, and approved the final damage and infertility (Aoki et al., 2005; García-Peiró et al., 2011; document. All authors contributed to the article and approved Nanassy et al., 2011). the submitted version. FUNDING CONCLUSION The authors acknowledge the support from the Ministry of In conclusion, long lysis treatment of porcine and bovine Science and Innovation, Spain (Grant No. AGL2017-88329-R), sperm is required for complete sperm DNA decondensation the Regional Government of Catalonia, Spain (Grant No. 2017- in these species. This is not necessary for human, horse SGR-1229), la Marató de TV3 (Grant No. 214/857-202039), and and donkey sperm, as lysis solutions used in previous University of Girona (Postdoc UdG-2020, to JR-M). studies led to complete DNA decondensation without any additional treatment. Moreover, we observed that the inclusion of proteinase K accelerates DNA decondensation. Thus, we SUPPLEMENTARY MATERIAL hypothesize that the species-specific differences in the resilience of sperm DNA to decondensation rely upon whether sperm DNA The Supplementary Material for this article can be found is condensed around protamine 1 and protamine 2, or around online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. protamine 1 only. 669182/full#supplementary-material REFERENCES Alkmin, D. V., Martinez-Alborcia, M. J., Parrilla, I., Vazquez, J. M., Martinez, E. A., and Roca, J. (2013). The nuclear DNA longevity in cryopreserved Adiga, S. 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This is an open-access article distributed under the terms of the Creative print]. Commons Attribution License (CC BY). The use, distribution or reproduction in Simon, L., Liu, L., Murphy, K., Ge, S., Hotaling, J., Aston, K. I., et al. (2014). other forums is permitted, provided the original author(s) and the copyright owner(s) Comparative analysis of three sperm DNA damage assays and sperm nuclear are credited and that the original publication in this journal is cited, in accordance protein content in couples undergoing assisted reproduction treatment. Hum. with accepted academic practice. No use, distribution or reproduction is permitted Reprod. 29, 904–917. doi: 10.1093/humrep/deu040 which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 April 2021 | Volume 9 | Article 669182