Theriogenology 185 (2022) 16e23 Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com Mild hypothermia and vitrification increase the mRNA expression of cold-inducible proteins in bovine oocytes and cumulus cells Jaume Gardela a, Mateo Ruiz-Conca a, Josune García-Sanmartín b, Alfredo Martínez b,  pez-Be Teresa Mogas c, Manel Lo  jar a, d, Manuel Alvarez-Rodríguez a, e, * a Department of Animal Health and Anatomy, Faculty of Veterinary Medicine, Universitat Autonoma de Barcelona, Bellaterra, Spain b ~ o, Spain Oncology Area, Center for Biomedical Research of La Rioja (CIBIR), Logron c noma de Barcelona, Bellaterra, Spain Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Universitat Auto d College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA, USA e €ping University, Division of Children's and Women Health (BKH), Obstetrics and Gynecology, Department of Biomedical and Clinical Sciences (BKV), Linko €ping, Sweden Linko a r t i c l e i n f o a b s t r a c t Article history: The cold-inducible RNA-binding protein (CIRBP) assists cells in adapting to new environmental condi- Received 22 November 2021 tions stabilizing specific mRNAs and promoting their translation. CIRBP participates in anti-apoptotic and Received in revised form anti-senescence processes, and its expression is induced by mild hypothermia, which may be advanta- 9 March 2022 geous to oocytes during vitrification. Several newly discovered small molecules, like zr17-2, mimic the Accepted 11 March 2022 Available online 18 March 2022 effects of cold temperatures by increasing the expression of CIRBP at normothermia. This study aimed to evaluate the mRNA changes of a group of cold-inducible protein-encoding and apoptotic genes in response to exogenous supplementation of zr17-2 (experiment 1) or CIRBP (experiment 2) in vitro Keywords: In vitro maturation matured bovine oocytes and their cumulus cells. In experiment 1, cumulus-oocyte complexes (COCs) Hypothermia mimetic were randomly exposed to three concentrations of zr17-2 (1, 10, and 100 mM) during 24 h of in vitro Cold-inducible RNA-binding protein maturation (IVM) under normothermia (38.5  C) or mild hypothermia (34  C) culture conditions. In Cow experiment 2, COCs were randomly exposed to three concentrations of CIRBP (2, 4, and 6 mg/mL) or subjected to mild hypothermia (34  C), followed by oocyte vitrification/warming after 20 h of IVM. The quantification of the selected gene transcript expression was performed separately in oocytes and cumulus cells by quantitative real-time PCR. We show that oocytes and cumulus cells exhibited similar mRNA expression responses to mild hypothermia and vitrification. However, minor differences were observed when COCs were exposed to exogenous supplementation with zr17-2 and CIRBP. In conclusion, the alterations observed in the mRNA expression in these experimental conditions may impact the quality of the cumulus-oocyte complexes in terms of vitrification and sublethal hypothermia challenges. In this sense, our results should complement other oocyte quality assessments for its application in future assisted reproductive techniques in the bovine species, including improving oocyte cryotolerance to vitrification. © 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction as the mitotic spindle [2e4]. During this process, the ultra-low temperatures affect mitochondrial activity, affecting apoptotic Oocyte cryopreservation is a valuable methodology for preser- pathways [5], inducing premature cortical granule extrusion, and ving genetic resources and contributes to developing assisted reducing successful fertilization chances [6]. reproductive techniques [1]. On the other hand, cryopreservation In mammalian cells, exposure to mild hypothermia (34  C) re- may damage the structure of the oocyte and chromosomes, as well duces the metabolism and the overall protein abundance, while the expression of a small group of homologous glycine-rich mRNA- binding proteins, known as cold-inducible proteins (CIPs), in- * Corresponding author. Department of Animal Health and Anatomy, Faculty of creases under this condition [7e12]. The CIPs bind to mRNA, acting Veterinary Medicine, Universitat Auto noma de Barcelona, Bellaterra, Spain. as regulatory molecules that play an essential role in RNA post-  E-mail address: manuel.alvarez.rodriguez@uab.cat (M. Alvarez-Rodríguez). https://doi.org/10.1016/j.theriogenology.2022.03.010 0093-691X/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 transcriptional control [13]. They are upregulated in response to 2. Materials and methods mild hypothermia and a variety of other cellular stressors, including osmotic stress, ultraviolet radiation, or hypoxia [14]. Unless otherwise stated, all chemicals and reagents were pur- The cold-inducible RNA-binding protein (CIRBP) is a constitu- chased from Sigma-Aldrich Co. (St. Louis, MO, USA). tively and ubiquitously expressed CIP considered a key component of the cellular response to moderate cold stress [15]. CIRBP has been identified in multiple tissues and species [16], including the bovine 2.1. Bovine COCs recovery and IVM ovary and testicle [17]. Although only a few body tissues are exposed to hypothermia in physiological conditions, the wide- Bovine ovaries were obtained from a local slaughterhouse spread expression of CIRBP [16] may suggest that it is likely (Escorxador de Sabadell, Barcelona, Spain) and transported to the involved in other relevant cellular processes at normothermia. laboratory at 35-37  C in 0.9% saline solution. The ovaries were Primarily located in the nucleus, CIRBP controls RNA transcription collected from cycling non-gravid heifers in follicular phase. and processing, while in the cytoplasm, CIRBP regulates mRNA Immature COCs were aspirated from ovarian follicles between 6 translation and turnover [8,18]. Eventually, CIRBP can be released and 8 mm in diameter using an 18-gauge needle attached to a low- into the extracellular space [19]. Once extracellular CIRBP is pre- pressure vacuum pump. Only COCs presenting more than three sent, it binds to toll-like receptor 4 (TLR4) and the myeloid differ- compact layers of cumulus cells and a homogeneous cytoplasm entiation factor 2 (MD2), activating several signaling pathways were selected for the study. The selected COCs were randomly including the NF-kB pathway [20,21]. The activation of such path- allocated in groups of 50 oocytes in 500 mL of maturation medium ways via CIRBP leads to an induction and release of proin- in 4-well dishes (Nalge Nunc International, Rochester, NY, USA) and flammatory cytokines and chemokines, including the IL-1b [19,22]. cultured following the experimental design. The maturation me- However, if IL-1b is modulated during the in vitro maturation (IVM) dium was composed of TCM199 with Earle's salts, L-glutamine, and of bovine cumulus-oocyte complexes (COCs) in the presence of sodium bicarbonate supplemented with 10% (v/v) fetal bovine CIRBP is still unknown. serum (FBS), 10 ng/mL epidermal growth factor, and 50 mg/mL Other CIPs, as the RNA-binding motif protein 3 (RBM3) and the gentamicin. serine and arginine-rich splicing factor 5 (SRSF5) [7,12], also bind to particular mRNAs, stimulating their translation, regulating their half-life, controlling their expression potential, and determining 2.2. Experiment 1: hypothermia mimetic zr17-2 supplementation their final functions [23,24]. Moreover, CIRBP and RBM3 bind to specific mRNAs involved in cell survival and anti-apoptotic cas- In experiment 1, COCs were randomly distributed in groups of cades [14], being potential candidates to improve oocyte cryotol- 50 oocytes in 4 experimental groups: exposed to control conditions erance to vitrification. Because oocyte vitrification can induce or exposed to three different concentrations of zr17-2 (1, 10, and apoptosis [25,26], the study of BCL2 (anti-apoptotic gene) [27], BAX 100 mM). These 4 experimental groups were incubated for 24 h in a (pro-apoptotic gene) [28], and its ratio is helpful to investigate the humidified atmosphere containing 5% CO2 at two different tem- effects of vitrification on oocytes and cumulus cells. perature conditions, 38.5  C or 34  C. After 24 h of IVM, COCs were A temporary increase in general adaptability generated by washed and denuded by gently pipetting (PIPETMAN ® P100L, controlled exposure to sub-lethal stressors has been used to Gilson, Spain) in phosphate buffer saline medium. Oocytes were improve cryotolerance in mammalian oocytes and embryos using harvested and the remaining media after denudation was centri- various strategies [29], such as heat stress [30] or high hydrostatic fuged (500g, 3 min, room temperature) to obtain the cumulus pressures [31]. However, little attention has been given to mild cells of the oocytes on each experimental group. The supernatant hypothermia as a sub-lethal stressor to enhance stress tolerance. In was discarded and the cryotube containing the cell pellet of each this sense, we have previously reported an increase in CIRBP group was immediately plunged in liquid nitrogen. Oocytes of each expression on bovine COCs exposed to mild hypothermia (33.5  C) group (n ¼ 25 oocytes/experimental group and replicate) were [32]. However, detrimental effects on oocyte nuclear and cyto- transferred to cryotubes using the minimum possible volume of plasmic maturation rates were reported due to the non-optimal media and directly immersed in liquid nitrogen. All samples were temperature conditions during the IVM [32]. Furthermore, some kept at 80  C until further analysis. The experiment was per- small molecules (including zr17-2) have been detected, capable of formed three times. mimicking the effect of hypothermia in the absence of cold stim- ulus [33], being good candidates for their application to improve cell cryotolerance. 2.3. Experiment 2: exogenous CIRBP supplementation In the present study, we hypothesized that oocytes and cumulus cells might modify the expression of selected genes during the IVM In experiment 2, COCs were randomly distributed in groups of in response to the exogenous supplementation with zr17-2 and 50 oocytes in 5 experimental groups: exposed to standard bovine CIRBP directly added to the culture medium. For this purpose, we IVM conditions (38.5  C, 5% CO2, in humidified atmosphere), aim to test the effect of exogenous supplementation with zr17-2 exposed to three different concentrations of exogenous CIRBP (2, 4, and CIRBP during the IVM of bovine COCs and whether this pro- and 6 mg/mL), or exposed to mild hypothermia (34  C, 5% CO2, in cedure implies upregulation of mRNA expression of the three CIPs humidified atmosphere). After 20 h of IVM, half of the COCs on each included (CIRBP, RBM3, and SRSF5), BCL2, and IL-1b, and down- experimental group were vitrified and ulteriorly warmed (n ¼ 25 regulation of BAX mRNA expression. In addition, we will determine oocytes/experimental condition and replicate). Vitrified/warmed the mRNA changes on three CIPs, apoptotic-related transcripts (BAX oocytes were returned for 4 h to the maturation dishes to complete and BCL2, and their ratio), and the pro-inflammatory interleukin 1b 24 h of IVM. After finishing the 24 h of IVM, COCs (n ¼ 25/experi- (IL-1b) induced by mild hypothermia and vitrification in both oo- mental group and replicate) were denuded by gently pipetting to cytes and cumulus cells. separate oocytes from cumulus cells, as before described. The experiment was performed three times. 17 J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 2.4. Oocyte vitrification and warming and the replicates as the random part of the LME. Pairwise com- parisons were adjusted by the Sidak test. After 20 h of IVM, COCs were partially denuded by gentle pipetting until a few layers of cumulus cells remained. Following a 3. Results modified vitrification protocol [34], oocytes were transferred into an equilibrium solution (TCM199 with HEPES supplemented with 3.1. Experiment 1: hypothermia mimetic zr17-2 supplementation 20% (v/v) FBS, 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (Me2SO)) for 9 min at room temperature (20- The relative mRNA expression of selected transcripts showed 22  C). After being equilibrated, oocytes were transferred into a that mild hypothermia induced most of the changes observed in vitrification solution (TCM199 with HEPES supplemented with 20% CIRBP, RBM3, SRSF5, BCL2, and BAX mRNA expression, both in oo- (v/v) FBS, 15% (v/v) EG, 15% (v/v) Me2SO, and 0.5 M sucrose) for 30 s. cytes (Fig. 1a) and cumulus cells (Fig. 1b). Groups of 5e6 oocytes were then picked up in a small volume of Mild hypothermia upregulated CIRBP and RBM3 mRNA expres- vitrification solution, placed on top of the polypropylene strip of a sion in oocytes and cumulus cells (p < 0.05). SRSF5 mRNA expres- Cryotop device (Kitazato Supply Co, Fujinomiya, Japan), and sion was upregulated in cumulus cells exposed to mild immediately plunged in liquid nitrogen. hypothermia culture conditions (p < 0.05), but no differences were For warming, the polypropylene strip of the Cryotop device was observed in oocytes. RBM3 mRNA expression was upregulated in immersed directly into TCM199 with HEPES supplemented with oocytes exposed to mild hypothermia at different concentrations of 20% (v/v) FBS containing 1 M sucrose at 37  C for 5 min. Recovered zr17-2 compared to the control group (p < 0.05). Besides, RBM3 oocytes were washed for 5 min in decreasing sucrose solutions mRNA expression was upregulated in oocytes exposed to mild (0.5 M, 0.25 M, and 0 M sucrose in TCM199 with HEPES). Once hypothermia at 100 mM zr17-2 compared to all the experimental warming was finished, oocytes were returned to the maturation groups at normothermia (p < 0.05). Additionally, RBM3 mRNA dishes allowing them to mature for four additional hours. expression was upregulated in oocytes exposed to mild hypother- mia at 10 mM zr17-2 compared to oocytes exposed to normo- 2.5. RNA extraction and cDNA synthesis thermia at 10 mM zr17-2 and IVM control conditions (p < 0.05). The mRNA expression of anti-apoptotic BCL2 was down- Total RNA from oocytes and cumulus cells was extracted using regulated in oocytes and cumulus cells cultured under mild hypo- the commercial miRNeasy Mini Kit (Qiagen, Barcelona, Spain) thermia conditions (p < 0.05), whereas mRNA expression of the following the manufacturer's instructions. The RNA concentration pro-apoptotic gene BAX was upregulated in cumulus cells cultured was determined using the Thermo Scientific NanoDropTM 2000 under mild hypothermia conditions (p < 0.05). (Fisher Scientific, Madrid, Spain). Synthesis of cDNA was performed When cultured at normothermia, the relative mRNA expression using the High-Capacity RNA-to-cDNA™ Kit (Fisher Scientific, of IL-1b was downregulated in oocytes by the 100 mM zr17-2 sup- Madrid, Spain) according to the manufacturer's instructions. The plementation compared to the control group (p < 0.05). cDNA obtained was stored at 20  C for subsequent analysis. 3.2. Experiment 2: exogenous CIRBP supplementation 2.6. Quantitative real-time PCR (qPCR) The relative mRNA expression of selected transcripts in Fig. 2 Data analysis was performed using the CFX Maestro software shows that mild hypothermia induced most of the changes version 3.3.3 (Bio-Rad Laboratories, Kabelsketal, Germany). Rela- observed in the CIRBP and RBM3 mRNA expressions, whereas the tive quantification of CIRBP, RBM3, SRSF5, BCL2, BAX, and IL-1b vitrification procedure induced changes in SRSF5 mRNA expression. transcripts was performed using the 2DDCt method [35] with CIRBP mRNA expression was upregulated in oocytes exposed to G3PDH as a housekeeping gene for normalization. Commercial mild hypothermia (vitrified and non-vitrified) compared to 6 mg/ gene-specific qPCR primers for bovine species were used (Pri- mL CIRBP (non-vitrified), 2 mg/mL CIRBP (vitrified and non- mePCR™SYBR® Green Assay, Bio-Rad Laboratories, Kabelsketal, vitrified), and vitrified control oocytes (p < 0.05). For cumulus Germany). The product sizes for each primer pair were confirmed cells, the CIRBP mRNA expression was upregulated in non-vitrified by loading the amplicons in an agarose gel using a gel imaging cells exposed to mild hypothermia compared to vitrified cumulus system (ChemiDoc XRS þ System, BioRad Laboratories, Inc., cells exposed to 2 mg/mL CIRBP (p < 0.05). Kabelsketal, Germany). RBM3 mRNA expression was upregulated in oocytes exposed to mild hypothermia (vitrified and non-vitrified) compared to the rest 2.7. Data analyses of the experimental groups (p < 0.05). In non-vitrified cumulus cells exposed to mild hypothermia, RBM3 mRNA expression was Each experiment was repeated three times. The relative mRNA upregulated compared to the rest of the non-vitrified experimental expression data of the genes included in the study were analyzed groups and the vitrified cumulus cells exposed to 2 and 6 mg/mL of by R software version 3.6.1 [36] with nlme [37] to develop linear exogenous CIRBP (p < 0.05). Additionally, RMB3 mRNA expression mixed-effects (LME) models and multcomp [38] to perform pair- was upregulated in vitrified cumulus cells exposed to mild hypo- wise comparisons. All data sets were analyzed for normal distri- thermia compared to the rest of vitrified and non-vitrified treat- bution and homoscedasticity using the ShapiroeWilk Normality ments, excluding the non-vitrified cumulus cells exposed to mild test and Levene's test, respectively. Non-normal distributed data hypothermia (p < 0.05). were transformed using the log(xþ1) transformation. The SRSF5 mRNA expression in oocytes and cumulus cells was threshold of significance was set at p < 0.05. increased by the vitrification procedure (p < 0.05). Besides, SRSF5 For experiment 1, the experimental group and the temperature mRNA expression was upregulated in vitrified oocytes exposed to during IVM (34  C or 38.5  C) were included as fixed effects and the mild hypothermia compared to non-vitrified oocytes exposed to 4, replicates as the random part of the LME. Pairwise comparisons 2 mg/mL CIRBP, and control conditions (p < 0.05). Additionally, were adjusted by the Sidak test. SRSF5 expression was upregulated in vitrified oocytes exposed to For experiment 2, the experimental group and vitrification 4 mg/mL CIRBP compared to non-vitrified oocytes exposed to 2 mg/ procedure (vitrified or non-vitrified) were included as fixed effects mL CIRBP and control conditions (p < 0.05). 18 J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 Fig. 1. Relative mRNA expression of selected genes in oocytes and cumulus cells of experiment 1. Relative mRNA expression ± SEM of CIRBP, RBM3, SRSF5, BCL2, BAX, and IL-1b in oocytes (a) and cumulus cells (b) of experimental groups in experiment 1. Supplementation with four concentrations of zr17-2 (0, 1, 10, 100 mM) incubated at 38.5  C or 34  C, 5% CO2 in humidified atmosphere. Different lowercase-letters represent statistical differences between experimental groups. Different uppercase-letters represent statistical differences between culture temperature conditions. The control group (0 mM zr17-2 incubated at 38.5  C, 5% CO2 in humidified atmosphere) was established as the reference group. Vitrification and mild hypothermia induced most of the changes (Fig. 3d). Besides, the ratio was increased in non-vitrified cumulus observed in the BCL2 mRNA of oocytes (Fig. 2a) and cumulus cells cells exposed to mild hypothermia compared to the rest of vitrified (Fig. 2b), respectively. In vitrified and non-vitrified cumulus cells cumulus cells (p < 0.05), except for the vitrified cumulus cells exposed to mild hypothermia, the BCL2 mRNA expression was exposed to mild hypothermia (Fig. 3d). downregulated compared to non-vitrified control cumulus cells and the vitrified and non-vitrified cumulus cells exposed to 4 mg/ 4. Discussion mL of exogenous CIRBP (p < 0.05). On the other hand, the vitrifi- cation procedure induced BAX and IL-1b mRNA expression changes Different strategies have been used to improve cryotolerance in in the cumulus cells. Additionally, BAX mRNA expression was mammalian oocytes by increasing the general adaptability induced upregulated in vitrified oocytes exposed to 6 mg/mL CIRBP by sublethal stressors [29]. Mild hypothermia, on the other hand, compared to vitrified oocytes exposed to mild hypothermia and has received little attention as an inductor of tolerance. Further- non-vitrified oocytes exposed to 4 mg/mL CIRBP (p < 0.05). more, the CIPs are potential candidates to mitigate the damage produced in oocytes during vitrification, mainly due to their 3.3. Apoptosis gene expression: BAX:BCL2 ratio involvement in cell survival and anti-apoptotic pathways [14]. This study has determined mRNA expression changes of CIPs and anti- The relative BAX:BCL2 ratios were increased by mild hypother- and pro-apoptotic transcripts in oocytes and cumulus cells induced mia in experiment 1 (Fig. 3a and b) and vitrification in experiment 2 by mild hypothermia, vitrification, and exogenous zr17-2 and CIRBP (Fig. 3c and d) (p < 0.05). supplementation. Additionally, in experiment 2, the ratio was increased in non- Oocytes and cumulus cells exhibited similar mRNA expression vitrified oocytes exposed to mild hypothermia compared to non- responses to mild hypothermia and vitrification, but minor differ- vitrified oocytes exposed to 4 mg/mL CIRBP and to control condi- ences were observed when COCs were exposed to exogenous tions (p < 0.05) (Fig. 3c). On the other hand, the ratio was increased supplementation with zr17-2 and CIRBP. Our findings may suggest in vitrified and non-vitrified cumulus cells exposed to mild hypo- a common underlying mechanism of such observed response to thermia compared to the non-vitrified treatments (p < 0.05) mild hypothermia and vitrification in both types of cells. The 19 J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 Fig. 2. Relative mRNA expression of selected genes related in oocytes and cumulus cells of experiment 2. Relative mRNA expression ± SEM of CIRBP, RBM3, SRSF5, BCL2, BAX, and IL-1b in oocytes (a) and cumulus cells (b) of treatments in experiment 2. Control (38.5  C, 5% CO2 in humidified atmosphere); three concentrations of exogenous CIRBP protein (2, 4, 6 mg/mL; 38.5  C, 5% CO2 in humidified atmosphere); and mild hypothermia (34  C, 5% CO2 in humidified atmosphere). Vitrified groups were represented with letter v. Different lowercase-letters represent statistical differences between treatments. Different uppercase-letters represent statistical differences between vitrified and non-vitrified groups. The control group was established as the reference group. bidirectional communication between oocytes and cumulus cells is transcription of neither both CIPs when cells were previously long essential to establish the optimal intrafollicular microenvironment, exposed to exogenous CIRBP and mild hypothermia. In contrast, which controls the growth and maturation of the follicles to pro- previous studies have reported an increase of CIRBP mRNA duce viable and competent oocytes [39,40]. In addition, this com- expression after oocyte vitrification [43,44], using the expression of plex communication may be involved in shared responses to ensure CIRBP as a marker of cryoinjury. Aside from its roles in hypother- that both cells quickly adapt to the new environmental conditions. mia, CIRBP has been shown to play key roles during early devel- This fact suggests that the mRNA expression changes that occurred opment [45] and cell protection against endogenous and external during the maturation of the oocyte may have potential effects stressors at normothermia [42]. Our study only analyzed an early during early embryo development [40]. In this sense, the regulation event during oocyte maturation and no later events on develop- of CIRBP expression in mature yak oocytes before vitrification may ment. Future research should focus on the effects of CIRBP and explain the improvement in its in vitro developmental competence other CIPs during in vitro early embryo development and tests its after vitrification/warming [41]. impact on embryo competence after oocyte vitrification/warming. Several CIPs have been previously identified; yet, two of them Another recently described RNA-binding protein induced by (CIRBP and RBM3) have been well-characterized since their dis- mild hypothermia and other stressors is SRSF5 [12]. SRSF5 belongs covery [7,8]. Both proteins regulate gene expression at the tran- to the serine-arginine rich splicing factor (SRSF) protein family, scription level by binding to different transcripts, allowing the cell which is involved in alternative splicing and mRNA processing [46]. to respond quickly to environmental changes [42]. Here, we Additionally, SRSF5 seems to perform specific functions in plurip- confirmed that the mRNA expressions of CIRBP and RBM3 were otent cells, managing the nuclear availability of splicing factors and significantly induced by mild hypothermia in oocytes and cumulus cytoplasmic activities by responding quickly to changing cellular cells, with or without an additional vitrification procedure. Even circumstances [47]. Our results showed that mild hypothermia though oocytes were exposed to ultralow temperatures during increased SRSF5 mRNA expression in cumulus cells but not in oo- vitrification, it seems that the brief exposure time to sub- cytes. Besides, SRSF5 mRNA expression was increased by the vitri- physiological temperatures was not enough to induce the fication procedure in both oocytes and cumulus cells. These results 20 J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 Fig. 3. Relative BAX:BCL2 ratios in oocytes and cumulus cells of experiment 1 and 2. Relative BAX:BCL2 ratio ± SEM in oocytes (a, c) and cumulus cells (b, d) of experiment 1 (a, b) and experiment 2 (c, d). Experiment 1: supplementation with four concentrations of zr17-2 (0, 1, 10, 100 mM) incubated at 38.5  C or 34  C, 5% CO2 in humidified atmosphere. Experiment 2: control (38.5  C, 5% CO2 in humidified atmosphere); supplementation with three concentrations of exogenous CIRBP protein (2, 4, 6 mg/mL; 38.5  C, 5% CO2 in humidified atmosphere); and mild hypothermia (34  C, 5% CO2 in humidified atmosphere). Vitrified groups were represented with letter v. Different lowercase-letters represent statistical differences between experimental groups. Different uppercase-letters represent statistical differences between culture temperature conditions (experiment 1) or vitrified and non-vitrified groups (experiment 2). The control group was established as the reference group for each experiment. contrast with what was observed in CIRBP and RBM3 mRNA molecules like IL-1b and the NF-kB pathway [22], and extracellu- expression, where only mild hypothermia was responsible for most larly, CIRBP acts as a damage-associated molecular signal [20]. of the changes. Although SRSF5 has been recently described as a Notably, IL-1b is a cytokine that plays a crucial role in inflammation, novel CIP [12], its mRNA expression responded differently from stimulating its production and other pro-inflammatory cytokines CIRBP and RBM3. [48,49]. Cytokines operate as mediators between the immune and The use of mild hypothermia sub-lethal stress and the vitrifi- reproductive systems and paracrine and autocrine ovarian regula- cation procedure lead to analyzing apoptotic-related transcripts in tory factors [50e52]. In addition to ovarian macrophages and this study. BCL2 is an anti-apoptotic gene that promotes cells sur- monocytes, other ovarian cell types can produce cytokines, like the vival by releasing mitochondrial cytochrome c [27], whereas BAX is granulosa cells [53]. Besides, the supplementation with IL-1b and the first pro-apoptotic member activated during apoptotic condi- tumor necrosis factor alpha stimulates the growth and maturation tions, accelerating cell death [28]. Other studies have investigated of bovine oocytes during in vitro culture [54]. Our study showed an the effect of vitrification on the expression of both genes [25,26]; upregulation of IL-1b mRNA expression in cumulus cells after however, the results have been inconsistent. Our results showed vitrification. However, our data cannot link the changes in IL-1b that mild hypothermia and vitrification affected BCL2 and BAX mRNA expression with the stimulation of the COCs growth after mRNA expression. Taken together, the detrimental effects caused warming. Further research is needed to test if IL-1b could stimulate by mild hypothermia and vitrification induced apoptotic pathways the COCs growth after warming to cope with the detrimental ef- in oocytes and cumulus cells were independent of the zr17-2 and fects of vitrification. Somehow, increased concentrations of zr17-2 CIRBP exogenous supplementation. Consistent with this conclu- exogenous supplementation trigger the downregulation of IL-1b sion, our BAX:BCL2 ratio data confirmed this hypothesis. mRNA expression. These results suggest a link between the two CIRBP has an important role in regulating inflammatory molecules, perhaps via a regulatory function of CIRBP accumulation 21 J. Gardela, M. Ruiz-Conca, J. García-Sanmartín et al. Theriogenology 185 (2022) 16e23 [22], but further studies should be performed to define the exact 10.1016/j.cryobiol.2012.09.001. [3] Chaves DF, Corbin E, Almin ~ ana C, Locatelli Y, Souza-Fabjan JMG, Bhat MH, et al. mechanism behind these results. Vitrification of immature and in vitro matured bovine cumulus-oocyte com- plexes: effects on oocyte structure and embryo development. Livest Sci 5. Conclusions 2017;199:50e6. https://doi.org/10.1016/j.livsci.2017.02.022. [4] Pitchayapipatkul J, Somfai T, Matoba S, Parnpai R, Nagai T, Geshi M, et al. Microtubule stabilisers docetaxel and paclitaxel reduce spindle damage and In conclusion, the current study has identified differences in the maintain the developmental competence of in vitro-mature bovine oocytes mRNA expression of bovine oocytes and cumulus cells exposed to during vitrification. Reprod Fertil Dev 2017;29:2028. https://doi.org/10.1071/ RD16193. mild hypothermia and vitrification. In addition, the exogenous [5] Zhang ZY, Yu XL, Cai MD, Liu YH, Liu JQ, Zhao SY, et al. Relationship between supplementation with zr17-2 to the maturation media has little bovine oocytes developmental competence and mRNA expression of impact on the CIPs mRNA expression of both oocytes and cumulus apoptotic and mitochondrial genes following the change of vitrification cells, but it affects IL-1b mRNA expression under physiological temperatures and cryoprotectant concentrations. Cryobiology 2020;97: 110e22. https://doi.org/10.1016/j.cryobiol.2020.09.009. temperatures and at the higher concentration used. Mild hypo- [6] Ghetler Y, Skutelsky E, Ben Nun I, Ben Dor L, Amihai D, Shalgi R. Human oocyte thermia induced most of the mRNA expression changes observed in cryopreservation and the fate of cortical granules. Fertil Steril 2006;86:210e6. this study. CIPs mRNA expression increased in response to mild https://doi.org/10.1016/j.fertnstert.2005.12.061. [7] Derry JMJ, Kerns JA, Francke U. RBM3, a novel human gene in Xp11.23 with a hypothermia. 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Mateo Ruiz- [19] Qiang X, Yang W-L, Wu R, Zhou M, Jacob A, Dong W, et al. Cold-inducible Conca: helped with the experiments and sample collection. Josune RNA-binding protein (CIRP) triggers inflammatory responses in hemorrhagic shock and sepsis. Nat Med 2013;19:1489e95. https://doi.org/10.1038/ García-Sanmartín: Supervision, helped with the experiments and nm.3368. sample collection. Alfredo Martínez: Supervision, Funding acqui- [20] Aziz M, Brenner M, Wang P. Extracellular CIRP (eCIRP) and inflammation. sition. Teresa Mogas: Supervision, Funding acquisition. Manel J Leukoc Biol 2019;106:133e46. https://doi.org/10.1002/JLB.3MIR1118-443R. Lo pez-Be  jar: Supervision, Funding acquisition. Manuel Alvarez- [21] Bolourani S, Sari E, Brenner M, Wang P. Extracellular CIRP induces an in- flammatory phenotype in pulmonary fibroblasts via TLR4. 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