Received: 7 March 2022 | Accepted: 24 April 2022 DOI: 10.1111/rda.14134 S H O R T C O M M U N I C AT I O N Changes in aquaporins mRNA expression and liquid storage at 17°C: A potential biomarker of boar sperm quality? Marina Castany Quintana1 | Jaume Gardela1,2 | Mateo Ruiz-­Conca1,2 | Manel López-­Béjar2,3 | Cristina A. Martinez1 | Heriberto Rodríguez-­Martinez1 | Manuel Álvarez-­Rodríguez1,2,4 1 Department of Biomedical and Clinical Sciences (BKV), Linköping University, Abstract Linköping, Sweden Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, 2 Department of Animal Health and Anatomy, Veterinary Faculty, Universitat which preserves sperm quality and ensures its fertilizing capacity. Routine quality Autònoma de Barcelona, Bellaterra, controls include the evaluation of sperm motility, viability and capacitation status. Barcelona, Spain 3 The physiological functions of all these features depend on transmembrane aqua- College of Veterinary Medicine, Western University of Health Sciences, Pomona, porins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made California, USA a relative quantification, using RT-­qPCR, of the mRNA of several sperm AQPs in AI-­ 4 Department of Animal Reproduction, liquid semen doses before and after a 48-­hr incubation period, aiming to determine INIA-­C SIC, Madrid, Spain possible quantitative compromising expression changes during the process that could Correspondence serve as a diagnostic tool. Our results showed a decrease in classical sperm motility Manuel Álvarez-­Rodríguez, Department of Animal Health and Anatomy, Veterinary variables (total and progressive motility and velocity) and sperm viability after 48-­hr Faculty, Universitat Autònoma de storage, whereas capacitation status increased overtime. mRNA expression increased Barcelona, Bellaterra, Barcelona, Spain. Emails: manuel.alvarez.rodriguez@uab.cat; in the orthodox AQP4 and AQP6 after 48-­hr incubation, relative to control (0 hr) and manualvro@gmail.com 24-­hr time-­points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 Funding information and AQP10 was higher after 48-­hr incubation, confirmed by AQP7-­protein valida- Ministerio de Ciencia, Innovación y tion using Western blot. Our results indicate that expression levels of AQPs-­mRNA Universidades; Svenska Forskningsrådet Formas can change in ejaculated pig spermatozoa under conditions of ex-­vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen. KEYWORDS AI doses, boar sperm, liquid storage, transcripts 1 | I NTRO D U C TI O N more marked as storage time increases (De Ambrogi et al., 2006). However, AI doses stored for more than 24 hr have been related In the swine industry, most artificial inseminations (AI) are carried to decreased fertility (Christensen et al., 2004). Unlike spermatozoa out using extended liquid semen as sperm cryopreservation re- in seminal plasma, which retain motility for a few hours, AI doses mains suboptimal, and with individual differences are becoming pursue to control not only the reduction of the metabolic activity by Rodríguez-­Martinez and Álvarez-­Rodríguez contributed equally as senior authors. This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made. © 2022 The Authors. Reproduction in Domestic Animals published by Wiley-­VCH GmbH. Reprod Dom Anim. 2022;00:1–4.  wileyonlinelibrary.com/journal/rda | 1 2 | CASTANY QUINTANA et al. lower temperatures (17°C) but also by providing the energy source viable (negative YO-­PRO-­1 fluorescence) and viable and capacitated and regulating pH, among other factors. (bright M540 fluorescence and negative YO-­PRO-­1 fluorescence). Aquaporins (AQPs), a family of ubiquitous transmembrane pro- teins, allow the transport of water and small molecules across the cell plasma membrane (Delgado-­Bermúdez et al., 2021). In mam- 2.2 | RNA isolation and qPCR analysis malian sperm, different AQPs have been identified being their main roles related to osmoadaptation and sperm motility activation after RNA was isolated using a commercial kit (RNeasy kit; Qiagen), ejaculation (Delgado-­Bermúdez et al., 2021). The group of orthodox following the manufacturer protocol, with slight modifications AQPs, exclusively permeable to water, includes AQP2, AQP4, AQP5, (Alvarez-­Rodriguez et al., 2020). Total RNA content, from 50 × 106 AQP6 and AQP8. In contrast, aquaglyceroporins, including AQP3, spermatozoa, and quality were determined by NanoDrop® 1000 AQP7, AQP9, AQP10 and AQP11, are also permeable to glycerol, (Thermo Fisher Scientific). Only samples with high RNA qual- urea and other small electrolytes. AQP3, AQP7 and AQP11 (Prieto-­ ity (260/280 nm absorbance ratio of 1.8–­2.2) were snap-­frozen in Martínez et al., 2016, 2017) and AQP9 (Vicente-­C arrillo et al., 2016) liquid nitrogen and stored at −80°C for analysis. The first-­strand have been identified in pig. AQP7 has been associated with sperm cDNA synthesis was performed using the High-­C apacity RNA-­to-­ motility in humans (Saito et al., 2004) but not in mice (Sohara cDNA Kit (Applied Biosystems), which consisted of 4 μg RNA in a et al., 2007). Thus, considering the function of AQPs and their influ- final volume of 20 μl. After the synthesis, the samples were stored at ence on pH through the regulation of water flow, this study aimed −20°C until further analyses. The Real-­Time PCR Detection System to track the changes in different AQPs during in vitro sperm liquid (CFX96; Bio-­Rad Laboratories, Inc) was used for the Quantitative storage at 17°C for up to 48 hr, and their correlation with sperm Polymerase Chain Reaction (qPCR). The reactions consisted of 0.5 μl quality parameters. of synthesized cDNA, 0.5 μl commercial gene-­specific primers for boar (PrimePCR SYBR® Green Assay: GAPDH, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP9, AQP10 and AQP11, Sus scrofa; Bio-­Rad 2 | M ATE R I A L S A N D M E TH O DS Laboratories, Inc), 5 μl of PowerUp SYBR Green Master Mix (Applied Biosystems) and water to a final volume of 10 μl. One cycle of uracil-­ The chemicals used in the experiments were of analytical grade. Unless DNA glycosylase (UDG) activation at 50°C for 2 min; one cycle of de- otherwise stated, all reagents were acquired from Sigma-­Aldrich. naturation at 95°C for 2 min; and 40 cycles of denaturation at 95°C for 5 s, annealing/extension at 60°C for 30 s, and a melting curve at 60–­95°C (0.5°C increments) for 5 s/step were used. Two technical 2.1 | Ejaculated spermatozoa replicates were used for each sample. The gene relative expression levels were quantified using the Pfaffl method (Pfaffl, 2001). The Boar ejaculates were supplied as commercial AI doses (10 batches of housekeeping gene GAPDH was used for cDNA normalization. 3-­boars per pool; Svenska Köttföretagen AB) extended in Hampshire Longlife (Durasperm; Kruuse) to 4.8 × 109 total spermatozoa/dose, each batch containing a pool of three different breeding, healthy, 2.3 | Western blotting for validation of AQP7 mature (1–­2 years old) boars of proven fertility and semen qual- protein expression ity (>80% motility, <15% total sperm abnormalities), and stored at 17°C, as recommended for AI purposes, for 0 hr (Control), 24 hr and Western blotting (WB) was performed following our previous pro- 48 hr, from the time of arrival to the laboratory (24 hr after sample tocol (Alvarez-­Rodriguez et al., 2021). Briefly, sperm samples were collection). homogenized by sonication in commercial lysis buffer (RIPA) at 4°C. Each AI dose was evaluated for motility (velocity and total Protein concentration was determined by the DC™ Protein Assay kit and forward progressive motility) using a light microscope (Zeiss) (Bio-­Rad). Each sample (25 µg) was denatured in 4× sample buffer equipped with a thermal plate (38°C), positive phase contrast optics and DTT for 10 min at 70°C, loaded into 4%–­20% SDS-­PAGE gels, (10× objective), a Charge Coupled Device (CCD) camera (UI-­1540LE-­ and transferred to polyvinylidene difluoride membranes. For protein M-­HQ, Ueye, IDS Imaging Development Systems GmbH) and the identification, membranes were blocked at room temperature for Qualisperm® Software (AKYmed). Two fluorescent probes in PBS 60 min and incubated overnight at 4°C with rabbit monoclonal anti-­ were combined to evaluate cell physiology: viability (YO-­ PRO-­ 1, AQP7 antibody (ab32826, Abcam; and MBS540430; MyBiosource) 75 nM; stains the nuclei of apoptotic or dead sperm) and capaci- at dilution 1/500. To standardize the results, a polyclonal IgG anti-­ tation status (Merocyanine 540, 2 µM; binds to membranes with GAPDH antibody (ab157156, Abcam) was used at a dilution 1/1,000 lower cholesterol:phospholipids ratio), after 15 min of incubation at in the same membranes. To visualize immunoreactivity, membranes 37°C in the dark. Analyses were performed using a fluorescence mi- were incubated 60 min at room temperature with secondary anti- croscope at 40× magnification (Eclipse Ti; Nikon). We recorded the body donkey anti-­rabbit IgGs (IRDye 800 CW-­labelled, 926-­32213, percentage of spermatozoa, in at least 4 random fields, identified as LI-­COR Biosciences, 238 Inc; Hamburg, The Netherlands) at dilution CASTANY QUINTANA et al. | 3 1/10,000. The membranes were scanned using the Odyssey CLx Total and progressive sperm motility and sperm viability de- (LICOR Biosciences). creased after 48-­hr storage and velocity dropped after 24-­hr in- cubation (Figure 1B). Capacitated-­viable spermatozoa increased in proportions after 24-­and 48-­hr incubation (Figure 1B). 2.4 | Statistical analysis All the AQPs had a significant negative correlation with total mo- tility, progressive motility and sperm viability. All AQPs studied, ex- Normal distribution and homoscedasticity of the data were ana- cept AQP6 and AQP10, presented a positive correlation with sperm lysed using the Shapiro–­W ilk normality test and Levene's test. capacitation (Figure S3). Non-­normal data distribution was restored using Log(x) transfor- mation prior to analysis. R-­version 3.6.1. (R Development Core Team, 2011) was used to conduct the statistical analyses, by linear 4 | D I S CU S S I O N A N D CO N C LU S I O N S mixed effects (LME) models and pairwise comparisons adjusted by Tukey's test. The threshold for significance was set at p < .05. Data To our knowledge, this is the first study to describe the differential ex- are presented as mean ± SEM, unless otherwise stated. Our LME pression of AQPs mRNA during liquid storage of boar AI doses at 17°C model included the incubation time as fixed effects and the pool up to 48 hr. Our results showed a decrease in sperm total motility at of samples as the random part of the model. Spearman's correla- 48 hr (72 hr after sample collection), in agreement with previous stud- tion analysis was performed among AQPs expressions and sperm ies describing a decrease in the total motile spermatozoa after 72-­hr quality parameters. storage (De Ambrogi et al., 2006; Khoi et al., 2021). Although these authors did not find any differences in sperm membrane integrity, our results showed a decrease after 48-­hr incubation, agreeing with a pre- 3 | R E S U LT S vious study (Dubé et al., 2004). Moreover, the capacitation status in our study increases after only 24-­hr incubation in a time-­dependent The mRNA expression of AQP3, 4, 6, 7 and 10 increased after 48 hr manner as has been previously suggested (Dubé et al., 2004). of storage (Figure 1A). In addition, mRNA from AQP4, 6, 7 and 10 AQP7, but not AQP9, has been previously described as a rel- increased after 24 hr of storage, relative to control (0 hr incuba- evant marker for non-­e mpirical studies on boar sperm handling tion) (Figure 1A). No differences were found in the rest of the AQPs (Vicente-­C arrillo et al., 2016). According to that statement, we measured (Figure S1). Moreover, AQP7 protein expression increases validated AQP7 mRNA results at the protein level. Our results after 48-­hr storage (p < .05), relative with control (1.00 ± 0.17), 24 hr confirm the increase in AQP7 mRNA and protein during storage, (1.02 ± 0.55) and 48-­hr storage (1.76 ± 0.37) (Figure S2). but not AQP9, the latter being positively related to sperm velocity F I G U R E 1 Relative AQPs mRNA expression changes (relative fold change) (A) and sperm quality parameters (B): total motility (TM), progressive motility (PM), velocity (Velocity), viability (Viab) and capacitation status (Capacitation), (n = 10) after 24-­and 48-­hr liquid storage at 17°C. Results are expressed in mean ± SEM. Different letters indicate differences among storage times 4 | CASTANY QUINTANA et al. and capacitation status. Also, as previously defined, AQP3 and of boar spermatozoa. International Journal of Andrology, 29(5), 543–­ AQP7 could be used as freezability biomarkers (Prieto-­Martínez 552. https://doi.org/10.1111/J.1365-­2605.2006.00694.X Delgado-­Bermúdez, A., Recuero, S., Llavanera, M., Mateo-­Otero, Y., Sandu, et al., 2016, 2017), which would be of high relevance in the por- A., Barranco, I., Ribas-­Maynou, J., & Yeste, M. (2021). Aquaporins are cine industry. Therefore, the increase in AQP3 and AQP7 mRNA in essential to maintain motility and membrane lipid architecture during our results might suggest a role to cope with the long-­t ime sperm mammalian sperm capacitation. Frontiers in Cell and Developmental liquid storage. Our results confront the main idea of spermatozoa Biology, 9, 1–­15. https://doi.org/10.3389/FCELL.2021.656438 Dubé, C., Beaulieu, M., Reyes-­Moreno, C., Guillemette, C., & Bailey, J. being considered a transcriptionally silent cell. However, this con- L. (2004). Boar sperm storage capacity of BTS and Androhep Plus: cept has been criticized recently, arguing that they could not be Viability, motility, capacitation, and tyrosine phosphorylation. completely quiescent cells (Santiago et al., 2021). Theriogenology, 62(5), 874–­886. https://doi.org/10.1016/J.THERI​ Overall, the differential cargo on several AQPs and, particularly, OGENO​LOGY.2003.12.006 Khoi, H. X., Shimizu, K., Yoneda, Y., Minagawa, I., Abe, Y., Kuwabara, Y., the AQP7 results at mRNA and protein level should lead to further Sasanami, T., & Kohsaka, T. (2021). Monitoring the reactive oxygen localization and functional level analysis for elucidating the exact species in spermatozoa during liquid storage of boar semen and its mechanism behind this AQPs expression changes. correlation with sperm motility, free thiol content and seasonality. Andrologia, 53(11), e14237. https://doi.org/10.1111/AND.14237 Pfaffl, M. W. (2001). A new mathematical model for relative quantifica- AC K N OW L E D G E M E N T S tion in real-­time RT-­PCR. Nucleic Acids Research, 29(9), e45. https:// This research was funded by the Research Council FORMAS, doi.org/10.1093/nar/29.9.e45 Stockholm (Projects 2017-­00946 and 2019-­00288), and by the Grant Prieto-­Martínez, N., Vilagran, I., Morató, R., Rivera del Álamo, M. M., PID2019-­ 108320RJ-­ I00 and IJCI-­ 2015-­ 24380 funded by MCIN/ Rodríguez-­Gil, J. E., Bonet, S., & Yeste, M. (2017). Relationship of AEI/10.13039/501100011033 (Spain) and FEDER funds (EU). To aquaporins 3 (AQP3), 7 (AQP7), and 11 (AQP11) with boar sperm resilience to withstand freeze–­thawing procedures. Andrology, 5(6), QualiSperm (AKYmed, Cheseaux-­sur-­Lausanne, Switzerland) for assist- 1153–­1164. https://doi.org/10.1111/andr.12410 ing with the computerized sperm motility and kinetics assessment. Prieto-­Martínez, N., Vilagran, I., Morató, R., Rodríguez-­Gil, J. E., Yeste, M., & Bonet, S. (2016). Aquaporins 7 and 11 in boar spermato- C O N FL I C T O F I N T E R E S T zoa: Detection, localisation and relationship with sperm quality. Reproduction, Fertility and Development, 28(6), 663–­672. https:// None of the authors have any conflict of interest to declare. doi.org/10.1071/RD14237 R Development Core Team (2011). R: A language and environment for sta- DATA AVA I L A B I L I T Y tistical computing. R Foundation for Statistical Computing, 1, 409. The data that support the findings of this study are available from https://doi.org/10.1007/978-­3-­540-­74686​-­7 Saito, K., Kageyama, Y., Okada, Y., Kawakami, S., Kihara, K., Ishibashi, K., the corresponding author upon reasonable request. & Sasaki, S. (2004). Localization of aquaporin-­7 in human testis and ejaculated sperm: Possible involvement in maintenance of sperm ORCID quality. The Journal of Urology, 172(5 Pt 1), 2073–­2076. https://doi. 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