Research in Veterinary Science 152 (2022) 181–189 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc The mRNA expression of the three major described cold-inducible proteins, including CIRBP, differs in the bovine endometrium and ampulla during the estrous cycle Jaume Gardela a, b, *, Mateo Ruiz-Conca a, b, Sergi Olvera-Maneu b, Manel López-Béjar b, c, Manuel Álvarez-Rodríguez a, b a Division of Children's and Women Health (BKH), Obstetrics and Gynecology, Department of Biomedical and Clinical Sciences (BKV), Linköping University, 58185, Linköping, Sweden b Department of Animal Health and Anatomy, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain c College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA A R T I C L E I N F O A B S T R A C T Keywords: The cold-inducible proteins (CIPs) are essential for post-transcriptional gene regulation playing diverse tissue- Cold-inducible RNA-binding protein specific roles in maintaining normal cellular function and morphogenesis. The potential implications of CIPs qPCR in reproductive events raise questions about their role in the physiology of the bovine reproductive tract. Western blot However, the expression changes of CIPs during the bovine estrous cycle have not been studied so far. Here, we Cattle hypothesized that the bovine estrous cycle could affect the mRNA expression of the CIPs and other candidate transcripts in the reproductive tract. This study aimed to examine estrous cycle-dependent mRNA expression patterns in the bovine endometrium and ampulla of three of the major described CIPs (CIRBP, RBM3, SRSF5), a set of inflammatory cytokines (IL-10, IL-18, IL-1β), and other candidate genes (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Endometrial and ampullar tissues were assessed by RT-qPCR. Additionally, the mRNA expression levels were correlated among them and with follicular progesterone and estradiol concen­ trations. The transcript levels of CIPs increased in the endometrium during stage III (Days 11–17) compared to stage I (Days 1–4) and IV (Days 18–20). In the ampulla, the mRNA expression of CIRBP increased during the late luteal phase (stage III), but no differences in the expression of other CIPs were observed. This study expands the current knowledge regarding mRNA expression in the endometrium and oviductal ampulla of cycling heifers, focusing mainly on the CIPs. A better understanding of the mechanisms within the uterus and oviduct during the estrous cycle is crucial to improving the fertility rate. 1. Introduction processing, localization, lifetime, and translation rate of specific mRNAs (Lunde et al., 2007). The RBPs are involved in several functions, During the bovine estrous cycle, different transcriptomic changes are including the modulation of inflammation and the immune system induced by hormonal regulation along the female reproductive tract in (Kafasla et al., 2014), contributing to different reproductive patho­ preparation for subsequent reproductive events (Forde et al., 2011). physiological conditions (Khalaj et al., 2017). Although inflammation Several gene regulatory processes are activated in response to a wide can cause pathological states in the female reproductive tract, an range of stressors to organize an appropriate cellular response (Harvey appropriate pro- and anti-inflammatory balance is required for repro­ et al., 2017). The post-transcriptional control of RNAs provides a critical ductive success (Cicchese et al., 2018). Besides, inflammation is crucial regulatory point in gene expression (Änkö, 2018). This regulation is during several physiological reproductive steps, including ovulation, achieved, in part, by the RNA-binding proteins (RBPs), which form RNA mating, embryo implantation (Jabbour et al., 2009), and the estrous ribonucleoproteins complexes when binding to RNA, defining the cycle (Bauersachs et al., 2005). However, the precise mechanism by * Corresponding author at: Department of Animal Health and Anatomy, Faculty of Veterinary Medicine, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain. E-mail address: jaume.gardela@uab.cat (J. Gardela). https://doi.org/10.1016/j.rvsc.2022.08.006 Received 3 December 2021; Received in revised form 8 July 2022; Accepted 6 August 2022 Available online 12 August 2022 0034-5288/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 which RBPs are modulated in the reproductive tissues during the bovine events (Bowman et al., 2000). estrous cycle is poorly understood. Revealing the mRNA changes of CIPs and inflammatory and A family of evolutionarily conserved RBPs named cold-inducible immune-related factors may help better understand the biological pro­ proteins (CIPs) (Ciuzan et al., 2015) is transcriptionally upregulated cesses in the endometrium and ampulla during the bovine estrous cycle. not only in response to mild hypothermia (Danno et al., 1997; Nish­ Here, we hypothesized that the mRNA expression of the CIPs is differ­ iyama et al., 1997; Tong et al., 2013; Rzechorzek et al., 2015; Fujita ently present through the bovine estrous cycle stages and in different et al., 2017), but also to different cellular stressors, such as UV radiation, regions of the female reproductive tract. Taking the ampulla and the osmotic stress, or hypoxia (Zhu et al., 2016). The cold-inducible RNA- endometrium as essential tissues for reproductive success in which binding protein (CIRBP) is a constitutively and ubiquitously expressed fertilization (Ellington, 1991) and implantation (Lee and DeMayo, 2004) CIP (Liao et al., 2017). Other CIPs are the RNA-binding motif protein 3 take place, respectively, we aimed to explore the mRNA expression of (RBM3) and the serine and arginine-rich splicing factor 5 (SRSF5), also three major described CIPs (CIRBP, RBM3, and SRSF5), inflammatory induced by mild hypothermia and other general stressors (Zhu et al., interleukins (IL-10, IL-1β, and IL-18), as well as other relevant immunity, 2016; Fujita et al., 2017). CIRBP is induced by mild hypothermia in cell proliferation, and apoptosis genes (IL-10RA, IL-10RB, BCL2, NLRP3, bovine cumulus cells (Gardela et al., 2019b), and it is localized in the STAT3, STAT5A, STAT6). bovine ovary (Gardela et al., 2019a) and the endometrium of women (Hamid et al., 2003). Several findings suggest that CIPs play various 2. Material and methods tissue-specific roles, including maintenance of normal cellular function and morphogenesis (Zhu et al., 2016). However, if the bovine estrous 2.1. Animals and tissue collection cycle modulates the mRNA expression of these CIPs along the female reproductive tract has not been determined. Commercial crossbreed beef postpubertal heifers (Bos taurus) aged Generally, CIRBP is mainly found in the nucleus but can be trans­ between 13 and 15 months were used for the recovery of the female located to the cytoplasm and released extracellularly under certain reproductive tracts (n = 20). Animals were slaughtered for commercial stress situations (Aziz et al., 2019). Intracellularly, CIRBP acts as an RNA purposes in a local slaughterhouse (Escorxador de Sabadell, Barcelona, chaperone regulating the mRNA stability of specific targets and trans­ Spain). The bovine reproductive tracts were classified into different mitting signals interacting with other proteins (Zhong and Huang, stages of the estrous cycle by the appearance of corpora lutea and antral 2017). Under stress, CIRBP can suppress apoptotic pathways promoting follicles according to the morphological classification described by cell survival (Liao et al., 2017), enhancing the expression of anti- Ireland et al. (Ireland et al., 1980), as follows: stage I, corresponding to apoptotic proteins like the B-cell lymphoma 2 (BCL2), which prevents the post-ovulatory phase, Days 1 to 4 (n = 6); stage II, corresponding to apoptosis by maintaining cell survival (Yang et al., 1997). the early luteal phase, Days 5 to 10 (not included due to the low number Extracellular CIRBP functions as a danger-associated molecular of animals found); stage III, corresponding to the late luteal phase, Days pattern (DAMP) response to promote inflammation (Qiang et al., 2013), 11 to 17 (n = 8); and stage IV, corresponding to the pre-ovulatory phase, being involved in diverse acute and chronic inflammatory diseases Days 18 to 20 (n = 6). Day 0 was considered as the day on which the (Zhong and Huang, 2017). Besides, CIRBP has been related to the female exhibits estrus (standing to be mounted) and other proceptive inflammasome (Yang et al., 2016). This multiprotein complex signaling phase behaviors (vocalization, mounting herd mates, and restlessness) platform that mediates immune responses activating inflammatory (Estill, 2015). Endometrial tissue samples were obtained exposing the caspases that facilitate the secretion of cytokines, which induce a pro- endometrial mucosa from intercaruncular areas at the base of the inflammatory form of cell death (Broz and Dixit, 2016). The protein uterine horn ipsilateral to the pre-ovulatory or freshly ovulated follicle, complex is formed in response to infection, damage, or other cellular <1 h from the harvest of the female reproductive tracts. Additionally, a stressors, containing a nucleotide-binding oligomerization domain-like section of the ampulla of the ipsilateral oviduct was obtained for each receptor (NLR) sensor molecule (Latz et al., 2013). The NLR pyrin animal. Only the epithelial folds on the inner surface of the ampulla domain containing 3 (NLRP3) inflammasome is a crucial mediator of were processed for RNA extraction. Immediately after tissue collection, inflammation in response to DAMPs, including cell-free DNA, extracel­ each sample was immersed in RNAlater™ stabilization solution (Fisher lular debris, or extracellular vesicles (Broz and Dixit, 2016; Shirasuna Scientific, Gothenburg, Sweden) and stored at − 80 ◦ C until RNA et al., 2020). The activation of the inflammasome complex triggers extraction. caspase 1, which activates the pro-inflammatory interleukins IL-1β and IL-18 (Latz et al., 2013). IL-1β mediates immune cell activation, cytokine 2.2. Follicular progesterone and estradiol concentrations production, and adhesion molecules, playing essential roles in several inflammatory diseases (Dinarello, 1996). IL-18 is a pro-inflammatory Follicular fluid was collected by aspiration of subordinate and cytokine first identified for its interferon-γ-inducing properties (Oka­ dominant follicles present in the ovaries using an 18 G needle coupled mura et al., 1998). with a 10-mL syringe. An individual pool of fluid from subordinate and As an anti-inflammatory cytokine, IL-10 is secreted by many cell dominant follicles from each ovary was used to determine follicular types, playing crucial roles in maintaining maternal immune tolerance concentrations of estradiol and progesterone. Samples were centrifuged (Bin and Sharma, 2015). The biological effects of IL-10 are exerted at 5000 X g for 10 min to discard cellular debris. Supernatants were through binding to the tetramer IL-10 receptor (IL-10R), with two recovered and stored at − 20 ◦ C until their analysis. ligand-binding subunits (IL-10R1 or IL-10RA) and two accessory Follicular progesterone and estradiol concentrations were deter­ signaling subunits (IL-10R2 or IL-10RB) (Bin and Sharma, 2015). mined by ELISA (Progesterone ELISA KIT and Estradiol ELISA KIT; Binding IL-10 to its receptor inhibits autophagy through the phosphoi­ Neogen Corporation, Ayr, UK). The absorbance values were obtained nositide-3 K (PI3K)-Akt signaling pathway (Park et al., 2011) and acti­ using a microplate reader (Sunrise-Basic Tecan; Tecan Austria GmbH, vates the Janus kinase (JAK)- signal transducer and activator of Grödig, Austria). According to the manufacturer, cross-reactivity of the transcription (STAT) pathway, which could activate STAT3 promoting progesterone ELISA antibody with other steroids was: deoxy­ various IL-10-responsive genes (Donnelly et al., 1999). The JAK-STAT corticosterone 2.5%, corticosterone 2.0%, and pregnenolone 2.0%. pathway regulates the transcription of multiple genes relevant to im­ Steroids with a cross-reactivity <2.0% are not presented. The cross- munity, development, and metabolic and stress responses (Verhoeven reactivity of the estradiol ELISA antibody with other steroids was et al., 2020). Activated by multiple cytokines (including interferons testosterone 1.0%. Steroids with a cross-reactivity <1.0% are not pre­ (INFs) and interleukins) (Bromberg, 2001), STATs are involved in sented. The precision within the test was assessed by calculating co­ several cell processes, and their dysregulation can induce pathological efficients of variation (CV, where CV = SD/mean × 100) from all 182 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 duplicate samples. The CV for progesterone and estradiol were 7.47% Homogenates were centrifuged (13,000 X g, 10 min, 4 ◦ C) and the su­ and 3.65%, respectively. A sensitivity of 0.23 ng/mL and 0.012 ng/mL pernatants were collected. Protein concentration was determined by the for progesterone and estradiol were respectively obtained. Finally, for detergent compatible (DC) protein assay (Bio-Rad Laboratories, Inc.; each pair of progesterone and estradiol data, a progesterone/estradiol Kabelsketal, Germany), using bovine serum albumin as standard. Then, (P4/E2) ratio was calculated. 25 μg of each sample were mixed with 4× sample buffer (NuPAGE LDS; Fisher Scientific, Gothenburg, Sweden) and 500 mM dithiothreitol. 2.3. RNA extraction and cDNA synthesis Samples were heated for 10 min at 70 ◦ C. Samples were run on 12–15% SDS-polyacrylamide gels. Odyssey® One-Color Protein Molecular Samples were homogenized in TRIzol using a bead mill (24 beats/s, Weight Marker (LI-COR Biosciences, Inc.; Hamburg, The Netherlands) 2 min, twice) (TissueLyser II with 7 mm stainless steel beads; Qiagen, was used as a molecular weight marker. For Western blot analysis, Sollentuna, Sweden) for total RNA extraction, as reported (Gardela proteins were transferred onto 0.2 μm polyvinylidene difluoride (PVDF) et al., 2020a). The homogenized was centrifuged (12,000 X g, 10 min, membranes (Fisher Scientific, Gothenburg, Sweden), previously acti­ 4 ◦ C) and the supernatant was retrieved and mixed with bromo­ vated with methanol. For protein identification, and after blocking chloropropane (100 μL/mL homogenized). After centrifugation (12,000 (Intercept Tris-buffered saline blocking buffer; LI-COR Biosciences, Inc.; X g, 15 min, 4 ◦ C), the aqueous phase was incubated with isopropanol Hamburg, The Netherlands) for 1 h at room temperature, membranes and RNA precipitation solution (1.2 M NaCl and 0.8 M Na2C6H6O7) were incubated overnight at 4 ◦ C with a rabbit monoclonal antibody (250 μL of each/500 μL aqueous phase). The mixtures were centrifuged against CIRBP (ab191885, Abcam, Cambridge, UK) at dilution 1:500. To (12,000 X g, 10 min, 4 ◦ C) and the supernatants were discarded. For each standardize the results, a rabbit monoclonal antibody against α-tubulin sample, 1 mL 75% ethanol was added. Supernatants were discarded (ab52866, Abcam, Cambridge, UK) was used at dilution 1:1000 in the after centrifugation (7500 X g, 5 min, 4 ◦ C), and the RNA pellets ob­ same membranes. To visualize immunoreactivity, membranes were tained were dried for 30 min in the fume hood. The RNA was dissolved incubated with secondary antibody donkey anti-rabbit IgGs (IRDye® in 30 μL of RNase-free water for 30 min on ice. 800 CW-labelled, 926–32,213, LI-COR Biosciences, Inc.; Hamburg, The After the extraction, the RNA concentration was determined using Netherlands). Then, PVDF membranes were scanned with Odyssey CLx the Thermo Scientific NanoDrop™ 2000 (Fisher Scientific, Gothenburg, Near-Infrared Fluorescence Imaging System (LI-COR Biosciences, Inc.; Sweden). The quality of the RNA was determined by the Agilent 2100 Hamburg, The Netherlands). Optical density was quantified by Image Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), using the Studio software version 5.2. (LI-COR Biosciences, Inc.; Hamburg, The samples with an RNA integrity number (RIN) value higher than 8. The Netherlands). cDNA synthesis was performed using the High-Capacity RNA-to-cDNA™ Kit (Fisher Scientific, Gothenburg, Sweden) according to the manufac­ 2.6. Statistical analyses turer's instructions. The reaction consisted of 5 μg RNA in a final volume of 50 μL. The cDNA obtained was stored at − 20 ◦ C for subsequent Data from qPCR analyses were exported with CFX Maestro™ 1.1 analysis. software version 4.1.2433.1219 (Bio-Rad Laboratories, Inc.; Kabelske­ tal, Germany) and data from Western blot analyses were exported with 2.4. Quantitative real-time PCR analyses Image Studio software version 5.2. (LI-COR Biosciences, Inc.; Hamburg, The Netherlands). All data sets were analyzed for normal distribution Following a previous protocol for quantitative real-time polymerase and homoscedasticity using the Shapiro–Wilk Normality test and Lev­ chain reaction (qPCR) (Gardela et al., 2020a) (CFX96™; Bio-Rad Lab­ ene's test, respectively. The log(x + 1) was used to transform non-normal oratories, Inc.; Kabelsketal, Germany), the mRNA relative expression distributions before analysis to achieve a normal distribution. Statistical levels were quantified using the 2-ΔΔCT method (Livak and Schmittgen, analyses were performed with R software version 3.6.1. (R Core Team, 2001). The reactions consisted of 2 μL cDNA, 250 nM of each primer, 2019). Data are presented as mean ± SEM. The threshold for signifi­ and 5 μL of PowerUp™ SYBR™ Green Master Mix (Applied Bio­ cance was set at P < 0.05. systems™, CA, USA) in a final volume of 10 μL. The PCR protocol Data from qPCR, Western blot, and follicular hormone analyses were consisted of one cycle of uracil-DNA glycosylase activation at 50 ◦ C for 2 analyzed using the nlme package (Pinheiro et al., 2020) to develop linear min; one cycle of denaturation at 95 ◦ C for 2 min; and 40 cycles of mixed-effects (LME) models and the multcomp package (Hothorn et al., denaturation at 95 ◦ C for 5 s, annealing/extension at 60.2 ◦ C for 30 s, 2008) to perform pairwise comparisons adjusted by Tukey's test. In the and a melting curve at 60–95 ◦ C (0.5 increments) for 5 s/step. Two LME model, the estrous cycle stages were included as the fixed factor technical replicates were performed per each sample and primer pair and the samples as the random part of the model. Data from qPCR an­ used. Four housekeeping genes were initially used for cDNA normali­ alyses were also analyzed using multiple Spearman's rank correlation zation (G3DPH, β-ACTIN, HPRT1, and TBP). After a preliminary analysis coefficients to explore the relationship between the mRNA expression of the results, the G3PDH was the most constantly expressed gene fold change of the target genes and between mRNA expression and through the tissues and treatments and was chosen for further analyses. follicular fluid hormone concentrations. Additionally, to investigate Commercial gene-specific qPCR primers for bovine were used (Pri­ whether the CIRBP protein abundance occurs in proportion to its mRNA mePCR™SYBR® Green Assay, Bio-Rad Laboratories, Kabelsketal, Ger­ relative levels, Spearman's rank correlation coefficients were performed. many). The specific sequences for each primer are registered by the company. The product sizes for each primer are shown in Supplemen­ 3. Results tary Table S1 and were confirmed by loading the amplicons in an agarose gel using a gel imaging system (ChemiDoc XRS+ System, Bio­ 3.1. Endometrial mRNA expression changes Rad Laboratories, Inc., Kabelsketal, Germany). The analysis indicated that the differences observed in the endo­ 2.5. Protein extraction and Western blot analyses metrium between stages of the estrous cycle in the mRNA expression of CIRBP, RBM3, SRSF5, STAT1, STAT5A, STAT6, IL-10RB, and NLRP3 Tissue fragments were homogenized in radioimmunoprecipitation were statistically significant (Fig. 1). The mRNA expression of the CIPs assay lysis buffer (RIPA; Fisher Scientific, Gothenburg, Sweden) sup­ (CIRBP, RBM3, and SRSF5) was higher expressed in stage III of the estrus plemented with 10 μL/mL protease inhibitors and ethyl­ cycle compared to stage I and IV (P < 0.05). Similarly, STAT1 and STAT6 enediaminetetraacetic acid (Thermo Scientific™ Halt™ Protease mRNA expressions were higher expressed in stage III of the estrous cycle Inhibitor Cocktail (100×); Fisher Scientific, Gothenburg, Sweden). compared to stage I and IV (P < 0.05). The STAT5A and NLRP3 mRNA 183 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 A B 30 10 a a 20 Relative mRNA expression Relative mRNA expression 8 a 10 6 a 4 b 5 a b b a a 2 b b b b b b b b 0 0 III III III IV IV III IV IV I I I I S- S- S- S- III III III IV IV IV I I I S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- CIRBP RBM3 SRSF5 STAT1 STAT3 STAT5A STAT6 C D 2.0 12 8 Relative mRNA expression Relative mRNA expression 1.5 4 1.0 a 2 a,b a 0.5 b a b 0.0 0 III IV I S- III III III III III III IV IV IV IV IV IV I I I I I I S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- BCL2 IL-10 IL-10RA IL-10RB IL-18 IL-1E NLRP3 Fig. 1. Endometrial relative mRNA expressions changes of the genes included in the study. Genes involved in (A) cold-inducible proteins, (B) factors of the signal transducer and activator of transcription (STAT) family, (C) the anti-apoptotic gene BCL2, and (D) inflammation, comparing stage I (S-I, n = 6), stage III (S-III, n = 8), and stage IV (S-IV, n = 6) of the bovine estrus cycle. Stage I was established as the reference group. Different letters show statistical differences between the bovine estrus cycle stages for the same gene (P < 0.05). Mean ± SEM. expressions were higher expressed in stages I and IV of the estrous cycle and STAT6 mRNA expressions were higher expressed in stage I of the compared to stage III (P < 0.05). The IL-10RB mRNA expression was estrous cycle compared to stage III (P < 0.05). higher expressed in stage III of the estrus cycle compared to stage I of the estrus cycle (P < 0.05). 3.3. Correlations between relative mRNA expression of genes and follicular hormone concentrations 3.2. Ampullary mRNA expression changes The follicular fluid progesterone and estradiol concentrations The analysis showed statistical differences in CIRBP, STAT1, STAT3, depicted a non-significant increasing trend as the estrous cycle pro­ STAT5A, STAT6, BCL2, IL-10RA, IL-10RB, and IL-1β mRNA expression gresses (Fig. 3A). The progesterone/estradiol (P4/E2) ratio did not differ between stages of the estrous cycle in the ampulla (Fig. 2). Of the three significantly between the stages of the estrous cycle (Fig. 3B). CIPs included in the study, only differences in the mRNA expression of The relative mRNA expression levels of the genes included in the CIRBP were found, increasing its mRNA expression in stage III of the study were correlated among themselves and with the follicular fluid estrus cycle compared to stage I and IV (P < 0.05). Similarly, STAT5A, concentrations for both tissues analyzed, namely the endometrium IL-10RA, and IL-10RB mRNA expressions were higher expressed in stage (Supplementary Fig. 1) and the ampulla (Supplementary Fig. 2). The III of the estrous cycle compared to stage I and IV (P < 0.05). Conversely, endometrial CIRBP mRNA expression positively correlates with several BCL2, STAT3, and IL-1β mRNA expression were lower expressed in stage mRNAs, independently of the stage of the estrous cycle. On the other III of the estrous cycle compared to stage I and IV (P < 0.05). The STAT1 hand, the ampullary CIRBP mRNA expression negatively correlates with 184 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 A B 4 8 a Relative mRNA expression a Relative mRNA expression 3 6 2 4 a a a b b a a,b 1 b a,b b 2 b b b 0 0 III III III III IV IV IV IV I I I I S- S- S- S- S- S- S- S- S- S- S- S- III III III IV IV IV I I I S- S- S- S- S- S- S- S- S- STAT1 STAT3 STAT5A STAT6 CIRBP RBM3 SRSF5 C D 1.5 5 a Relative mRNA expression Relative mRNA expression a a 4 1.0 3 a b 2 0.5 b b b b a a 1 b 0.0 0 III IV I S- III III III III III III IV IV IV IV IV IV I I I I I I S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- S- BCL2 IL-10 IL-10RA IL-10RB IL-18 IL-1E NLRP3 Fig. 2. Ampullary relative mRNA expressions changes of the genes included in the study. Genes involved in (A) cold-inducible proteins, (B) factors of the signal transducer and activator of transcription (STAT) family, (C) the anti-apoptotic gene BCL2, and (D) inflammation, comparing stage I (S-I, n = 6), stage III (S-III, n = 8), and stage IV (S-IV, n = 6) of the bovine estrus cycle. Stage I was established as the reference group. Different letters show statistical differences between the bovine estrus cycle stages for the same gene (P < 0.05). Mean ± SEM. interleukins and their receptors, and with NLRP3 during stage III. Spearman correlation coefficient of 0.56 in the ampulla implied that 56% of the variation in CIRBP protein expression was explained by 3.4. CIRBP protein expression in the bovine endometrium and ampulla known mRNA abundances. Differences in relative CIRBP protein expression between stages of 4. Discussion the estrous cycle were evidenced by Western blot analysis of both in the endometrial and ampullary tissues (Fig. 4). In the endometrium, the The RBPs have key functions in the post-transcriptional regulation of CIRBP protein expression was higher expressed in stage III compared to mRNA, being crucial in tissue homeostasis and pathophysiology in stage I (P < 0.05). In the ampulla, the CIRBP protein expression was multiple tissues, including the female reproductive tract (Khalaj et al., higher expressed in stage III compared to stage IV (P < 0.05). 2017; Änkö, 2018). Although the implications of RBPs in the repro­ ductive process are poorly understood, CIPs may have potential impli­ 3.5. CIRBP protein abundance in proportion to CIRBP mRNA levels cations in early reproductive events during the peri-ovulatory phase (Gardela et al., 2020b). The present study demonstrates that the mRNA In both endometrial and ampullary tissues, the CIRBP protein of CIPs and other candidate factors are differentially expressed in the expression correlated with the abundances of its corresponding mRNA, endometrium and ampulla during the bovine estrous cycle. but not strongly (Fig. 5). The Spearman correlation coefficient of 0.61 in It has been hypothesized that the regular cyclic changes induced the endometrium implied that 61% of the variation in CIRBP protein during the estrous cycle in the bovine female reproductive tract may expression was explained by known mRNA abundances. Similarly, the lead to changes in the mRNA expression of three CIPs: CIRBP, RBM3, and 185 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 A B 250 20 200 15 150 ng/mL 10 100 5 50 0 0 IV IV IV I III I III I III S- S- S- S- S- S- S- S- S- Progesterone Estradiol Ratio Fig. 3. Follicular fluid hormone concentrations and ratio progesterone/estradiol. (A) Follicular fluid progesterone and estradiol concentrations (ng/mL) and (B) ratio progesterone/estradiol during stage I (S-I, n = 6), stage III (S-III, n = 8), and stage IV (S-IV, n = 6) of the bovine estrus cycle. Non-significant differences between stages were found. Mean ± SEM. A D-tubulin - 50 kDa Stage I CIRBP - 19 kDa C D-tubulin - 50 kDa 5 Stage III Relative protein CIRBP expression CIRBP - 19 kDa a 4 D-tubulin - 50 kDa Stage IV CIRBP- 19 kDa 3 a,b B 2 a b a,b D-tubulin - 50 kDa Stage I CIRBP - 19 kDa 1 b D-tubulin - 50 kDa Stage III 0 CIRBP - 19 kDa III III IV IV I I S- S- S- S- S- S- D-tubulin - 50 kDa Endometrium Ampulla Stage IV CIRBP - 19 kDa Fig. 4. Relative CIRBP protein expression in endometrium and ampulla. Western blot analyses of relative CIRBP protein expression in (A) the endometrium and (B) ampulla. (C) Relative protein expression of CIRBP in the endometrium and the ampulla, comparing stage I (S-I, n = 6), stage III (S-III, n = 8), and stage IV (S-IV, n = 6) of the estrus cycle. Stage I was established as the reference group. Different letters show statistical differences between the estrus cycle stages for each tissue (P < 0.05). Mean ± SEM. SRSF5. However, as multifunctional proteins that contribute to the was increased in the ampulla during stage III of the estrous cycle, which maintenance of normal cellular function (Zhu et al., 2016), we aimed to was also confirmed by results obtained at the protein level. This situa­ explore other candidate-related factors. Our results showed increased tion resembles that reported for the human uterus, whose CIRBP protein the CIRBP, RBM3, and SRSF5 mRNA expression in the endometrium levels in the endometrium are strongly increased during the secretory during the late luteal phase (stage III). Only CIRBP relative mRNA level (luteal) phase and inversely correlated to the proliferative activity in 186 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 A B Relative CIRBP mRNA expression Relative CIRBP mRNA expression rho = 0.61 15 p < 0.01 rho = 0.56 10 p < 0.01 10 5 5 0 0 0 2 4 6 0.0 0.5 1.0 1.5 2.0 Relative CIRBP protein expression Relative CIRBP protein expression Fig. 5. Correlation between relative CIRBP mRNA and protein expression. Spearman rank correlation scatter plots and linear regression with its 95% confidence interval (gray area) of the relative CIRBP mRNA expression and the CIRBP protein expression (A) in the endometrium and (B) ampulla. endometrial glands (Hamid et al., 2003). These results may be related to NLRP3 has been linked with the preparation of the bovine endometrium the described activities of CIRBP in the regulation of post-transcriptional during early implantation (Suzuki et al., 2018), our results might suggest and translational events, cell proliferation, and protection against that, in the absence of pregnancy, the increase of NLRP3 mRNA apoptosis, among other molecular and cellular activities (Zhu et al., expression could lead to the preparation of the endometrium for the 2016). However, the exact mechanism behind these results needs early reproductive events. further research to be clarified. We analyzed the factors of the STAT family that can activate gene High progesterone levels in the luteal phase can induce apoptotic- transcription, transducing signals from the cell membrane into the nu­ like regression of granulosa cells (Zhang et al., 2021). However, cleus (Sadowski et al., 1993). Here, we reported differential and oppo­ apoptosis is less important in the oviduct changes during the estrous site STAT1, STAT3, STAT5A, and STAT6 mRNA expressions during the cycle (Steffl et al., 2008). As shown here, we demonstrate differential bovine estrous cycle in the endometrium and ampulla. expression of the anti-apoptotic gene BCL2 and its correlations with Our results showed an increase in the endometrium STAT1 mRNA other genes during the bovine estrous cycle in the ampulla. The mRNA expression during the late luteal phase, being the STAT1 one of the first expression of BCL2 increased during the pre- and post-ovulatory phases STAT proteins detected in the INF signal transduction pathways, which (stage IV and I, respectively), showing a common positive correlation involves, among others, stimulation of apoptosis (Bromberg, 2001). with the mRNA expression of CIRBP, SRSF5, IL-1β, and IL-18, and a Previous studies have shown that, in the event of gestation, the STAT1 negative correlation with the mRNA expression of IL-10. Our data agree transcript increases in the endometrium during the period of maternal with the proposed defense mechanism to protect the ampulla from recognition of pregnancy (Day 16) and then amplifies at implantation damage caused by apoptosis during the luteal phase (Zhang et al., 2021). (Day 20) (Carvalho et al., 2016). In the ampulla, the STAT1 mRNA IL-1β stimulates the infiltration and migration of immune cells to the expression was increased during the post-ovulatory phase compared to bovine oviduct epithelium (Nakamura et al., 2021). In the present study, the late luteal phase. This dual modulation of STAT1 mRNA opens new the mRNA expression of IL-1β was significantly higher in the ampulla questions to understand the biological functions and specific mechanism during pre-ovulatory and post-ovulatory phases compared with the late of STAT1 during the bovine estrous cycle. luteal phase. Similar results were reported in the bovine endometrium, The activation of STAT3, by multiple cytokines or other factors, such in which the mRNA expression of IL-1β increased around ovulation as hormones (Verhoeven et al., 2020), is crucial for wound healing compared to the luteal phase (Fischer et al., 2010). Other studies (Chang et al., 2004), restoration of tissue integrity (Sano et al., 1999), demonstrate the induction of inflammatory cytokines and acute-phase and the resolution of the immune response (Fielding et al., 2008). Our proteins in the bovine endometrium during post-partum (Gabler et al., results showed a decrease in STAT3 transcript in the ampulla during the 2010; Pothmann et al., 2021), first inflammatory immune responses late luteal phase, suggesting that STAT3 signaling in the ampulla may be (Gärtner et al., 2016), and endometritis (Wagener et al., 2017). How­ involved in early reproductive events, like ovulation, sperm transport, or ever, our data did not show IL-1β mRNA expression changes in the fertilization. As for the STAT6, previous studies have associated STAT6 endometrium, probably due to the variation of mRNA expression be­ with puberty onset (Nguyen et al., 2018) and age at first corpus luteum tween animals. As previously proposed in the bovine endometrium (Fortes et al., 2011). As shown here, STAT6 mRNA expression increased (Fischer et al., 2010), we suggest a similar functional IL1 system in the in the ampulla during the pre- and post-ovulatory phases, contrary to bovine ampulla. what happened the endometrium. These results may suggest possible Our results did not show any changes in IL-10 mRNA expression roles of STAT6 during early reproductive events, yet not fully either in the endometrium or ampulla during the bovine estrous cycle. understood. However, we demonstrated higher IL-10RA and IL-10RB mRNA Our study gives a first approach to understanding the modulation of expression in the ampulla during the late luteal phase, and higher IL- the CIPs and related transcripts during the bovine estrous cycle. 10RB mRNA expression in the endometrium during the late luteal phase Regarding the classification of the reproductive tracts, the methodology that may suggest a role of these receptors in the homeostasis mainte­ applied has been used for over 40 years to assign animals into stages and nance in that specific stage of the estrus cycle. the length of such stages; however, the lack of complete analysis to Several immune-mediated processes contribute to the regulation of confirm that classification should be considered, and the presence of reproductive physiology (Yockey and Iwasaki, 2018) and may be animals in different hormonal profiles and physiological statuses should beneficial for subsequent fertility at the time of insemination (Wagener not be discarded. Moreover, further studies are needed to link the et al., 2021). Our results revealed higher NLRP3 mRNA expression in the different correlation patterns between follicular hormones and mRNA endometrium during the pre- and post-ovulatory phases. As long as the expression levels observed during the bovine estrous cycle in this study. 187 J. Gardela et al. Research in Veterinary Science 152 (2022) 181–189 5. Conclusions Bauersachs, S., Ulbrich, S.E., Gross, K., Schmidt, S.E.M., Meyer, H.H.D., Einspanier, R., Wenigerkind, H., Vermehren, M., Blum, H., Sinowatz, F., et al., 2005. Gene expression profiling of bovine endometrium during the oestrous cycle: detection of In summary, we have revealed significant changes in the mRNA molecular pathways involved in functional changes. J. Mol. Endocrinol. 34 (3), expression during the bovine estrous cycle. Here, we reported an in­ 889–908. crease in the mRNA and protein expression of CIRBP during the late Bin, Cheng S., Sharma, S., 2015. Interleukin-10: a pleiotropic regulator in pregnancy. Am. J. Reprod. Immunol. 73 (6), 487–500. luteal phase (Days 11–17) in both the endometrium and ampulla of Bowman, T., Garcia, R., Turkson, J., Jove, R., 2000. STATs in oncogenesis. Oncogene 19 cycling heifers. Additionally, we demonstrated changes in the mRNA (21), 2474–2488. expression of other CIPs (RBM3 and SRSF5), a pro-inflammatory cyto­ Bromberg, J.F., 2001. Activation of STAT proteins and growth control. BioEssays 23 (2), 161–169. kine (IL-1β), and other related genes involved in inflammatory pathways Broz, P., Dixit, V.M., 2016. Inflammasomes: mechanism of assembly, regulation and (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Our signalling. Nat. Rev. Immunol. 16 (7), 407–420. data provide novel molecular insights into the mRNA changes during the Carvalho, A.V., Eozenou, C., Healey, G.D., Forde, N., Reinaud, P., Chebrout, M., Gall, L., Rodde, N., Padilla, A.L., Delville, C.G., et al., 2016. 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The interleukin-10 signal transduction pathway and regulation of gene expression in mononuclear phagocytes. (Spain) and FEDER funds (EU). JG was supported by the Government J. Interf. Cytokine Res. 19 (6), 563–573. of Catalonia (Generalitat de Catalunya, AGAUR) co-financed with the Ellington, J.E., 1991. The bovine oviduct and its role in reproduction: a review of the European Social Found (2018FI_00236) and was awarded with an in­ literature. Cornell Vet. 81 (3), 313–328. Estill, C.T., 2015. Initiation of puberty in heifers. In: Hopper, R.M. (Ed.), Bovine ternational mobility grant from Col⋅legi Oficial de Veterinaris de Bar­ Reproduction. First Edit. Wiley Balckwell, Oxford, UK, pp. 195–202. celona (COVB, Beques Ramon Turró). MRC is supported by the Fielding, C.A., McLoughlin, R.M., McLeod, L., Colmont, C.S., Najdovska, M., Grail, D., Government of Spain, Ministry of Education, Culture and Sports Ernst, M., Jones, S.A., Topley, N., Jenkins, B.J., 2008. IL-6 regulates neutrophil trafficking during acute inflammation via STAT3. J. Immunol. 181 (3), 2189–2195. (Training programme for Academic Staff FPU15/06029) and Ministry of Fischer, C., Drillich, M., Odau, S., Heuwieser, W., Einspanier, R., Gabler, C., 2010. Universities (Mobility grant for short stays and temporary transfers for Selected pro-inflammatory factor transcripts in bovine endometrial epithelial cells beneficiaries of the FPU program; EST19/00848). are regulated during the oestrous cycle and elevated in case of subclinical or clinical endometritis. Reprod. Fertil. Dev. 22 (5), 818–829. Forde, N., Beltman, M.E., Duffy, G.B., Duffy, P., Mehta, J.P., Ó’Gaora, P., Roche, J.F., CRediT authorship contribution statement Lonergan, P., Crowe, M.A., 2011. Changes in the endometrial transcriptome during the bovine estrous cycle: effect of low circulating progesterone and consequences for conceptus elongation. Biol. Reprod. 84 (2), 266–278. Jaume Gardela: Conceptualization, Methodology, Software, Vali­ Fortes, M.R.S., Reverter, A., Nagaraj, S.H., Zhang, Y., Jonsson, N.N., Barris, W., dation, Formal analysis, Investigation, Data curation, Writing – original Lehnert, S., Boe-Hansen, G.B., Hawken, R.J., 2011. A single nucleotide draft, Visualization. Mateo Ruiz-Conca: Methodology, Investigation, polymorphism-derived regulatory gene network underlying puberty in 2 tropical Writing – review & editing. Sergi Olvera-Maneu: Methodology, Vali­ breeds of beef cattle. J. Anim. Sci. 89 (6), 1669–1683. Fujita, T., Hiroaki, Higashitsuji, Hisako, Higashitsuji, Liu, Y., Itoh, K., Sakurai, T., dation, Investigation, Writing – review & editing. Manel López-Béjar: Kojima, T., Kandori, S., Nishiyama, H., Motoi, Fukumoto, et al., 2017. TRPV4- Conceptualization, Resources, Writing – review & editing, Supervision, dependent induction of a novel mammalian cold-inducible protein SRSF5 as well as Funding acquisition. Manuel Álvarez-Rodríguez: Conceptualization, CIRP and RBM3. Sci. Rep. 7 (1), 2295. Gabler, C., Fischer, C., Drillich, M., Einspanier, R., Heuwieser, W., 2010. Time-dependent Resources, Writing – review & editing, Supervision, Project adminis­ mRNA expression of selected pro-inflammatory factors in the endometrium of tration, Funding acquisition. primiparous cows postpartum. Reprod. Biol. Endocrinol. 8 (1), 152. Gardela, J., García-Sanmartín, J., Ruiz-Conca, M., Álvarez-Rodríguez, M., Martínez, A., Mogas, T., López-Béjar, M., 2019a. Immunohistochemical identification of CIRBP in Declaration of Competing Interest bovine ovary and testicle. Anim. Reprod. 16 (3), 727. Gardela, J., Ruiz-Conca, M., Álvarez-Rodríguez, M., Mogas, T., López-Béjar, M., 2019b. Induction of CIRBP expression by cold shock on bovine cumulus–oocyte complexes. None. Reprod. Domest. Anim. 54 (S4), 82–85. Gardela, J., Jauregi-Miguel, A., Martinez, C.A., Rodriguez-Martinez, H., Lopez-Bejar, M., Alvarez-Rodriguez, M., 2020a. Semen modulates the expression of NGF, ABHD2, Acknowledgements VCAN, and CTEN in the reproductive tract of female rabbits. Genes (Basel) 11 (7), 758. The authors are grateful for the staff from the Escorxador de Sabadell Gardela, J., Ruiz-Conca, M., Martinez, C.A., Wright, D., López-Béjar, M., Rodriguez- Martinez, H., Alvarez-Rodriguez, M., 2020b. The expression of cold-inducible RNA- slaughterhouse (Sabadell, Barcelona, Spain), and for the infrastructure binding protein mRNA in sow genital tract is modulated by natural mating, but not support from Linköping University, including genomics core facility. We by seminal plasma. Int. J. Mol. Sci. 21 (15), 5333. also thank Amaia Jauregi Miguel, for her kind assistance with the qPCR Gärtner, M.A., Peter, S., Jung, M., Drillich, M., Einspanier, R., Gabler, C., 2016. 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