International Journal of Molecular Sciences Article The Expression of Cold-Inducible RNA-Binding Protein mRNA in Sow Genital Tract Is Modulated by Natural Mating, but not by Seminal Plasma Jaume Gardela 1,2 , Mateo Ruiz-Conca 1,2 , Cristina A. Martinez 1 , Dominic Wright 3 , Manel López-Béjar 2,4 , Heriberto Rodriguez-Martinez 1 and Manuel Alvarez-Rodriguez 1,2, * 1 Department of Biomedical and Clinical Sciences (BKV), Division of Children’s and Women Health (BKH), Obstetrics and Gynaecology, Linköping University, 58185 Linköping, Sweden; jaume.gardela@uab.cat (J.G.); mateo.ruiz@uab.cat (M.R.-C.); cristina.martinez-serrano@liu.se (C.A.M.); heriberto.rodriguez-martinez@liu.se (H.R.-M.) 2 Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; manel.lopez.bejar@uab.cat or manel.lopezbejar@westernu.edu 3 Department of Physics, Chemistry and Biology, Faculty of Science and Engineering, Linköping University, 58185 Linköping, Sweden; dominic.wright@liu.se 4 College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA * Correspondence: manuel.alvarez-rodriguez@liu.se; Tel.: +46-(0)729427883  Received: 10 June 2020; Accepted: 24 July 2020; Published: 27 July 2020  Abstract: The RNA-binding proteins (RBPs), some of them induced by transient receptor potential (TRP) ion channels, are crucial regulators of RNA function that can contribute to reproductive pathogenesis, including inflammation and immune dysfunction. This study aimed to reveal the influence of spermatozoa, seminal plasma, or natural mating on mRNA expression of RBPs and TRP ion channels in different segments of the internal genital tract of oestrous, preovulatory sows. Particularly, we focused on mRNA expression changes of the cold-inducible proteins (CIPs) and related TRP channels. Pre-ovulatory sows were naturally mated (NM) or cervically infused with semen (Semen-AI) or sperm-free seminal plasma either from the entire ejaculate (SP-TOTAL) or the sperm-rich fraction (SP-AI). Samples (cervix to infundibulum) were collected by laparotomy under general anaesthesia for transcriptomic analysis (GeneChip® Porcine Gene 1.0 ST Array) 24 h after treatments. The NM treatment induced most of the mRNA expression changes, compared to Semen-AI, SP-AI, and SP-TOTAL treatments including unique significative changes in CIRBP, RBM11, RBM15B, RBMS1, TRPC1, TRPC4, TRPC7, and TRPM8. The findings on the differential mRNA expression on RBPs and TRP ion channels, especially to CIPs and related TRP ion channels, suggest that spermatozoa and seminal plasma differentially modulated both protein families during the preovulatory phase, probably related to a still unknown early signalling mechanism in the sow reproductive tract. Keywords: transcriptomics; microarrays; spermatozoa; seminal plasma; cold-inducible proteins; cold-sensitive TRP ion channels; pig 1. Introduction Mammals reproduce by internal fertilization releasing billions of spermatozoa into the female reproductive tract to achieve maximum chances of fertilization, despite only thousands reaching the oviduct [1], where fertilization takes place. Ejaculate deposition into the female reproductive tract affects the molecular and cellular function of reproductive organs proximal and distal to the insemination site [2]. The interaction between the female reproductive tract and semen induces mRNA Int. J. Mol. Sci. 2020, 21, 5333; doi:10.3390/ijms21155333 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5333 2 of 21 and protein expression changes in endometrial and oviductal tissues modulating important processes for reproduction, such as angiogenesis, sperm storage and selection, oviduct contractility and oocyte transportation, and preimplantation embryo development, previously reported in pigs [3–6] and other species [7–11]. Many RNA-binding proteins (RBPs) are involved in the control of inflammation and immune dysfunction, which can contribute to pathogenesis of reproductive disorders [12]. Each step of RNA metabolism (transcription, splicing, polyadenylation, stabilization, edition, capping, or translation) is assisted by the RBPs [13]. These proteins are the most important regulators of RNAs and crucial for gene regulation, containing different structural RNA-binding motifs or domains, such as RNA recognition motif (RRM), K homology domain, double-stranded RNA binding domain, or zinc fingers, forming ribonucleoprotein complexes by binding double or single-stranded RNA [14]. Beyond their RNA-binding domains, the RBPs hold many structural modules that facilitate protein-protein interactions and catalytic events; thereby, they are incorporated in numerous intracellular processes [14]. The cold-inducible proteins (CIPs) are evolutionarily conserved RBPs that have been detected in multiple organisms from different taxa [15]. The cold-inducible RNA-binding protein (CIRBP, also known as CIRP, or heterogenous nuclear ribonucleoprotein A18, hnRNP A18), the RNA-binding motif protein 3 (RBM3), and the serine and arginine-rich splicing factor 5 (SRSF5, also known as SRp40) are CIPs transcriptionally up-regulated in response to moderately low temperatures and other cellular stressors, such as hypoxia, DNA damage, or osmotic stress [16,17]. CIRBP was first described in the late 1990s as a novel RBP which expression was induced in mouse cells in response to cold stress [18]. Since this discovery, investigations revealed its role in many cellular stress responses, such as mRNA stability, cell survival, proliferation, stress adaptation, and tumor formation [19]. Intracellularly, CIRBP acts as a RNA chaperone to facilitate translation, whereas extracellularly, it seems to play a crucial role in the promotion of inflammation and injury [20,21]. Both CIRBP and RBM3 are structurally similar to the heterogeneous nuclear ribonucleoproteins and belong to the glycine-rich RBP family class IVa [15], which contains, at the N-terminal end, one conserved RRM and two ribonucleoprotein domains (RNPs), known as RNP1 and RNP2 [17]. The C-terminal contains a less conserved arginine-glycine-rich (RGG) domain, and this is the reason why CIRBP and RBM3 belong to the large family of glycine-rich proteins [17]. SRSF5 belongs to the serine/arginine-rich family protein (also called serine/arginine-rich splicing factors, SRSFs), characterized by the presence of one or two N-terminal RRMs followed by a downstream domain rich in arginine and serine residues [22]. Heterogeneous nuclear ribonucleoproteins and SRSFs are both active factors involved in the regulation of pre-mRNA splicing, and due to its derived origin from a common ancestor, have been suggested to play an important role in the evolution of the alternative splicing mechanism [23]. Through alternative splicing, a single gene can encode for multiple variants, greatly enhancing the transcriptome complexity, the variety of proteins and hence, the diverse biological functions [24]. The induction of CIPs is dependent on transient receptor potential (TRP) V4, V3, and M8 ion channel proteins [16,25]. The TRP mammalian proteins can be classified in four major families [26]: the ankyrin-related TRP channel family (TRPA), the TRPC family (classical or canonical), the melastatin- related TRP channel family (TRPM), and the channels homologous to vanilloid receptor (TRPV). These receptors are non-selective cation channels permeable for Ca2+ that open in response to multiple chemical and physical stimuli [27–29], providing Ca2+ influx pathways [26]. Most of them have thermosensitive abilities, like TRPM8, TRPA1, and TRPC5, which have been proposed as cold-sensitive ion channels [29]. Even though current research has focused on discerning the effects produced by spermatozoa and seminal plasma (SP) in endometrium and oviductal mRNA expression in the sow reproductive tract [30–32], to the best of our knowledge, there is a lack of literature regarding the differential mRNA expression levels of genes encoding for RBPs and TRP ion channels. Here, we specifically studied the differential mRNA expression levels of CIPs and related TRP ion channels in the internal genital Int. J. Mol. Sci. 2020, 21, 5333 3 of 21 tract of pre-ovulatory sows induced by the interaction of semen (spermatozoa and/or SP) with the genital mucosa. 2. Results The presence of inflammation was analyzed by the mRNA expression of the interleukin-8, also called CXCL8, a chemokine induced in lipopolysaccharide-stimulated monocytes inducing neutrophil migration [33] and often used as classical inflammation marker. In this study, mRNA expression of CXCL8 was down-regulated (p < 0.05) by natural mating (NM) in the cervix (fold change −4.38) and the proximal uterus (fold change −3.06), and down-regulated (p < 0.05) by artificial insemination with the sperm-peak fraction (Semen-AI) in the utero-tubal junction (fold change −5.78), whereas no differences were found in the rest of the tissues and treatments. Spermatozoa were only present in the utero-tubal junction of NM and Semen-AI treated animals but not in animals treated with sperm-free SP or controls. 2.1. Induced mRNA Expression Changes on RBPs and TRP Ion Channels Table 1 displays the mRNA expression levels of the 39 differentially expressed genes (DEGs) encoding for RBPs and TRP ion channels for each treatment and tissue sample. The number of DEGs (up- and down-regulated, p < 0.05), with respect to the control group (cervical infusion with Int. J. Mol. 5021,mL Sci. 2020, x Beltsville thawing solution, BTS), that codify for RBP family 7 of 23 containing the RRM domain (IPR000504) were displayed in Figure 1a. Even though the statistical analyses did not The number of DEGs (up‐ and down‐regulated, p < 0.05), with respect to the control group determine overall (cervicaldifferences infusion with among groups 50 mL Beltsville in the thawing number solution, BTS),of up-regulated that DEGs codify for RBP family encoding for RBPs containing (NM: 16 genes,theSemen-AI: RRM domain6(IPR000504) genes, sperm-free SP infusion were displayed from in Figure 1a. Eventhe sperm-peak though fraction the statistical analyses (SP-AI): did 8 genes, not determine overall differences among groups in the number of up‐regulated DEGs encoding for and sperm-free SP infusion from the whole ejaculate (SP-TOTAL): 9 genes; p > 0.05), the infundibulum RBPs (NM: 16 genes, Semen‐AI: 6 genes, sperm‐free SP infusion from the sperm‐peak fraction (SP‐ showed a greater number AI): 8 genes, of up-regulated and sperm‐free SP infusionDEGs from theencoding for (SP‐TOTAL): whole ejaculate 9 genes; pp><0.05), RBPs (12 genes, 0.05)thecompared to the proximalinfundibulum uterus (2 genes), showed awithin all treatments. greater number Sperm-containing of up‐regulated DEGs encoding for RBPs treatments (12 genes, pdown-regulated < 0.05) a compared to the proximal uterus (2 genes), within all treatments. Sperm‐containing treatments greater number of DEGs encoding for RBPs (NM: 24 genes, and Semen-AI: 21 genes; p < 0.01) compared down‐regulated a greater number of DEGs encoding for RBPs (NM: 24 genes, and Semen‐AI: 21 to sperm-freegenes; SP treatments p < 0.01) compared(SP-AI: 3 genes, to sperm‐free SPand SP-TOTAL: treatments 1 gene). (SP‐AI: 3 genes, and SP‐TOTAL: 1 gene). ✱ Up-regulated DEGs Up-regulated DEGs ✱ vx t th f t TJ p vx t t TJ f th p tU In U tU U In m m Is Is ox ox C C U U is is A A D Pr D Pr Down-regulated DEGs Down-regulated DEGs ✱✱✱ ✱✱ x Ut Ut J th p f t t vx TJ th p f In tU U In UT Cv Am m Is Is st ox ox C U is A Di Pr Pr D Figure 1. Heatmap Figure 1.composition of the number Heatmap composition of differentially of the number of differentiallyexpressed genes expressed genes < 0.05) (p <(p0.05) in thein the porcine porcine female female reproductive tractreproductive tract 24 h post‐treatment. 24 h post-treatment. Genes belonging Genes belonging to: (a) to: (a) the the RNA‐binding RNA-binding protein family protein family that contain the RNA recognition motif domain (IPR000504) in their structure, and (b) that contain the RNA recognition motif domain (IPR000504) in their structure, and (b) the transient the transient receptor potential ion channels, up‐regulated (brown) and down‐regulated (blue). Cvx: receptor potential ion channels, cervix; DistUt: up-regulated distal uterus; (brown) ProxUt: proximal andutero‐tubal uterus; UTJ: down-regulated (blue). junction, Isth: Cvx: isthmus; Amp:cervix; DistUt: distal uterus;ampulla; ProxUt: Inf: proximal infundibulum. uterus; NaturalUTJ: utero-tubal mating: sows mated junction, with a boar;Isth: isthmus; Semen‐AI: Amp: ampulla; Inf: sows artificially inseminated with the sperm‐peak portion extended to 50 mL with Beltsville thawing solution (BTS); infundibulum. Natural mating: sows mated with a boar; Semen-AI: sows artificially inseminated with SP‐AI: sows cervically infused with the sperm‐free seminal plasma (SP) from pooled sperm‐peak the sperm-peak portion portion (50 mL);extended SP‐TOTAL: tosows 50 mL withinfused cervically Beltsville thawing with the solution sperm‐free SP of the(BTS); SP-AI: (50 whole ejaculate sows cervically infused with mL). the sperm-free All treatmentsseminal plasma were compared (SP) with from(cervical controls pooledinfusion sperm-peak portion with 50 mL (50 mL); SP-TOTAL: BTS). Asterisks represent statistical differences between treatments and tissues (* p < 0.05, ** p < 0.01, *** p < 0.001). sows cervically infused with the sperm-free SP of the whole ejaculate (50 mL). All treatments were compared with controls Figure 1b displays (cervical infusion the number with(up‐ of DEGs 50 mL BTS). Asterisksp represent and down‐regulated, statistical < 0.05) that codify differences for TRP between ion treatments channels. A and tissues greater (* p < number of 0.05, DEGs** p < 0.01, encoding for*** TRP < 0.001). p ion channels were up‐regulated by NM (9 genes, p < 0.05) compared to SP‐TOTAL (0 genes). The down‐regulated DEGs encoding for TRP ion channels were significantly affected by NM (14 genes, p < 0.001) compared to the rest of the treatments (Semen‐AI: 4 genes, SP‐AI: 1 gene, and SP‐TOTAL: 2 genes). The number of common DEGs that codify for RBPs and TRP ion channels (up‐ and down‐ regulated, p < 0.05) in the reproductive tract of sows between treatments were displayed in a series of Venn diagrams [34], indicating which DEGs were identified as common to each treatment per tissue (Figure 2). As may be seen, the mRNA expression pattern was affected by the entrance of the Int. J. Mol. Sci. 2020, 21, 5333 4 of 21 Table 1. Differential mRNA expression (p < 0.05) encoding for RNA-binding proteins and transient receptor potential ion channels in the different segments of the sow reproductive tract (cervix to infundibulum), 24 h post-treatment. Natural Mating Semen-AI SP-AI SP-TOTAL Gene Symbol UP DOWN UP DOWN UP DOWN UP DOWN Cvx (1.62) UTJ (1.27) CIRBP * - - - - - - - Amp (1.41) Inf (1.25) Amp (−1.91) RBM11 - - - - - - Inf (−1.73) RBM15B ProxUt (1.19) - - - - - - - Cvx (−1.38) RBM18 - Amp (−1.28) - Cvx (−1.31) - - - Cvx (−1.22) Inf (−1.26) ProxUt (1.18) RBM19 - - - - - DistUt (−1.15) - Isth (1.24) RBM24 - Isth (−1.76) - - Isth (1.22) - - - RBM25 - - - - - - Inf (1.25) - RBM26 - - - - DistUt (1.11) - - - Amp (−1.53) RBM34 - - Inf (−1.18) Inf (1.19) - UTJ (1.49) - Inf (−1.23) RBM38 Cvx (1.27) - - - - UTJ (−1.33) - - RBM39 - - - - DistUt (1.2) - - - RBM42 - - - Inf (−1.17) - Inf (−1.16) - - RBM44LOC100627002 - UTJ (−1.2) UTJ (1.39) - - - - - RBM47 DistUt (1.18) UTJ (−1.39) - - - - Cvx (1.21) UTJ (1.17) UTJ (1.24) RBM5 Isth (1.18) - Inf (1.21) - - - - Isth (1.15) Amp (1.21) Inf (1.52) Int. J. Mol. Sci. 2020, 21, 5333 5 of 21 Table 1. Cont. Natural Mating Semen-AI SP-AI SP-TOTAL Gene Symbol UP DOWN UP DOWN UP DOWN UP DOWN DistUt (−1.4) ProxUt (−1.35) Cvx (−1.41), UTJ (−2) ProxUt RBM7 - - - - - - Isth (−2.48) (−1.33), Inf Amp (−1.98) (−1.5) Inf (−2.09) Isth (−1.23) RBM8A - Inf (−1.35) - - - - - Inf (−1.25) RBMS1; - DistUt (−1.23) - - - - - - LOC102159432 Cvx (1.41) Amp (1.71) Isth (1.3) RBMS2 ProxUt (−1.41) - - - - - Inf (1.74) Amp (1.5) Inf (1.4) RBMX - Isth (−1.18) - - - - Inf (1.33) - SRSF1 - Amp (−1.36) - Amp (−1.31) - - Inf (1.43) - Isth (−1.6) SRSF12; Amp (−1.46) DistUt (1.25) - Amp (−1.53) - - - - LOC100739399 Inf (−1.66) Inf (−1.68) UTJ (−1.26) SRSF3; Amp (−1.51) Isth (−1.28) LOC100523801; - - - – Inf (1.5) - Inf (−1.27) Amp (−1.48) LOC106508591 Inf (−1.24) SRSF4 - - - - DistUt (1.14) DistUt (1.16) - SRSF5; UTJ (1.28) - - Cvx (−1.21) Inf (1.19) - - - LOC100623241 * UTJ (−1.25) SRSF7 - - - Isth (−1.26) - - Inf (1.61) - Amp (−1.38) Int. J. Mol. Sci. 2020, 21, 5333 6 of 21 Table 1. Cont. Natural Mating Semen-AI SP-AI SP-TOTAL Gene Symbol UP DOWN UP DOWN UP DOWN UP DOWN Cvx (−2.46) TRPA1; DistUt (−7.73) DistUt - - - - - - LOC100627895 * ProxUt (−6.34) (−2.47) Isth (−1.14) TRPC1 Inf (1.52) ProxUt (−1.43) - - - - - - TRPC3 UTJ (1.71) - UTJ (1.65) UTJ (1.64) - - - ProxUt (2.23) TRPC4 - - - - - - - Inf (1.29) Cvx (−2.29) DistUt (−2.44) ProxUt (−3.31) ProxUt ProxUt TRPC5 * - UTJ (−2.65) - - ProxUt (−1.86) - (−1.57) (−1.39) Isth (−1.78) Amp (-1.84) Inf (−3.21) TRPC7 Inf (1.1) - - - - - - - UTJ (1.74) TRPM3 - - - - - - Amp (−1.68) Isth (3.37) DistUt (2.13) TRPM6 Inf (−1.25) - - DistUt (1.55) - - - ProxUt (2.2) DistUt (1.24) TRPM7 - - - - - - Isth (1.12) TRPM8; - Isth (−1.23) - - - - - LOC100627434 * TRPV1 - - Inf (−1.38) - - - - TRPV2 - - Inf (−1.05) - - - - TRPV6 - - Cvx (2.32) - - - - - Cvx: cervix; DistUt: distal uterus; ProxUt: proximal uterus; UTJ: utero-tubal junction; Isth: isthmus; Amp: ampulla; and Inf: infundibulum. UP: up-regulation and DOWN: down-regulation (in bold). The fold change for each treatment and tissue is presented between brackets. Asterisks mark genes that codify for RNA-binding proteins included in the cold-inducible proteins and genes that codify for cold-sensitive transient receptor potential ion channels. Natural mating: sows mated with a boar; Semen-AI: sows artificially inseminated with the sperm-peak portion extended to 50 mL with Beltsville thawing solution (BTS); SP-AI: sows cervically infused with the sperm-free seminal plasma (SP) from pooled sperm-peak portion (50 mL); SP-TOTAL: sows cervically infused with the sperm-free SP of the whole ejaculate (50 mL). All treatments were compared with controls. Int. J. Mol. Sci. 2020, 21, 5333 7 of 21 Figure 1b displays the number of DEGs (up- and down-regulated, p < 0.05) that codify for TRP ion channels. A greater number of DEGs encoding for TRP ion channels were up-regulated by NM (9 genes, p < 0.05) compared to SP-TOTAL (0 genes). The down-regulated DEGs encoding for TRP ion channels were significantly affected by NM (14 genes, p < 0.001) compared to the rest of the treatments (Semen-AI: 4 genes, SP-AI: 1 gene, and SP-TOTAL: 2 genes). The number of common DEGs that codify for RBPs and TRP ion channels (up- and down-regulated, p < 0.05) in the reproductive tract of sows between treatments were displayed in a series of Venn diagrams [34], indicating which DEGs were identified as common to each treatment per tissue (Figure 2). As may be seen, the mRNA expression pattern was affected by the entrance of the early sperm-rich fraction of the ejaculate in the female reproductive tract (Figure 2a). The common DEGs between NM and Semen-AI groups tended to be down-regulated (12 down-regulated DEGs vs. 4 up-regulated DEGs, Figure 2a). The combination between NM and SP-TOTAL groups only demonstrated common down-regulated DEGs in the uterus (Figure 2b). Combinations including sperm-freeInt. J.SP Mol.treatments Sci. 2020, 21, x showed common up- and down-regulated DEGs generally observed 8 of 23 in the uterus (Figure 2b–d). However, the combination between Semen-AI and SP-AI groups showed uterus (Figure 2b–d). However, the combination between Semen‐AI and SP‐AI groups showed common up- and down-regulated DEGs in the oviduct (Figure 2c). The combination between NM and common up‐ and down‐regulated DEGs in the oviduct (Figure 2c). The combination between NM Semen-AIand groups Semen‐AI showedgroups a predominant numbernumber showed a predominant of up-ofand up‐down-regulated and down‐regulated DEGs DEGsininthe theoviductal segmentsoviductal compared to the segments uterinetosegments compared (11 DEGs the uterine segments (11in oviductal DEGs tissues in oviductal vs.vs.55 DEGs tissues DEGs inin uterine segments, uterine Figure segments, Figure 2a). the 2a). Curiously, Curiously, the proximal proximal uterus uterus showed showed a common a common down‐regulation ofof TRPC5 down-regulation TRPC5 expression in all combinations. Additionally, the utero‐tubal junction showed a common up‐ expression in all combinations. Additionally, the utero-tubal junction showed a common up-regulation regulation of TRPC3 between NM and Semen‐AI groups, and SP‐AI and Semen‐AI groups (Figure of TRPC32a,c). between NM and Semen-AI groups, and SP-AI and Semen-AI groups (Figure 2a,c). Venn2.diagrams Figure 2.Figure of commonly differentially expressed genes (p < 0.05) in the porcine female Venn diagrams of commonly differentially expressed genes (p < 0.05) in the porcine female reproductive tract codifying for RNA-binding reproductive tract codifying for RNA‐bindingproteins proteins andand transient transient receptor receptor potential potential ion channels ion channels (up- or down-regulated), 24 h post-treatment. (up‐ or down‐regulated), 24 h post‐treatment.(a)(a)Sows Sows mated witha boar mated with a boar (Natural (Natural mating, mating, orange)orange) or sows artificially artificially inseminated or sows inseminated with thewith the sperm‐peak sperm-peak portion portion extended extended to to 5050mLmL withBeltsville with Beltsville thawing solution thawing solution (BTS) (Semen‐AI, green); (b) Natural mating (orange) or sows cervically infused (BTS) (Semen-AI, green); (b) Natural mating (orange) or sows cervically infused with the with the sperm‐free SP of the whole ejaculate (50 mL) (SP‐TOTAL, blue); (c) Semen‐AI (green) sows sperm-free SP of the whole ejaculate (50 mL) (SP-TOTAL, blue); (c) Semen-AI (green) sows cervically cervically infused with the sperm‐free seminal plasma (SP) from pooled sperm‐peak portion (50 mL) infused with (SP‐AI, purple); (d) SP‐AIseminal the sperm-free plasma (purple) or SP‐TOTAL(SP)(blue). fromCvx: pooled cervix,sperm-peak portion ProxUt: proximal uterus,(50 mL) (SP-AI, DistUt: purple); (d) SP-AI distal uterus,(purple) or SP-TOTAL UTJ: utero‐tubal (blue). junction, Amp: Cvx: and ampulla, cervix, ProxUt: proximal Inf: infundibulum. uterus, The acronyms DistUt: distal of genes common uterus, UTJ: to treatment utero-tubal junction, (crossing per tissueAmp: sectors) are ampulla, identified. and Inf: infundibulum. The acronyms of genes common to treatment per tissue (crossing sectors) are identified. 2.2. mRNA Expression Changes on CIPs and Related TRP Ion Channels Of the total number of DEGs encoding for RBP family included in the study (26 genes, Table 1), only two genes belong to the CIPs: CIRBP and SRSF5 [16,17]. The NM group up‐regulated CIRBP mRNA expression in the cervix, utero‐tubal junction, ampulla, and infundibulum (p < 0.05), and up‐ regulated SRSF5 mRNA expression in the utero‐tubal junction (p < 0.05), whereas Semen‐AI down‐ regulated SRSF5 mRNA expression in the cervix (p < 0.05) and SP‐AI up‐regulated SRSF5 mRNA Int. J. Mol. Sci. 2020, 21, 5333 8 of 21 2.2. mRNA Expression Changes on CIPs and Related TRP Ion Channels Of the total number of DEGs encoding for RBP family included in the study (26 genes, Table 1), only two genes belong to the CIPs: CIRBP and SRSF5 [16,17]. The NM group up-regulated CIRBP mRNA expression in the cervix, utero-tubal junction, ampulla, and infundibulum (p < 0.05), and up-regulated SRSF5 mRNA expression in the utero-tubal junction (p < 0.05), whereas Semen-AI down-regulated SRSF5 mRNA expression in the cervix (p < 0.05) and SP-AI up-regulated SRSF5 mRNA expression in the infundibulum (p < 0.05). Of the total number of DEGs encoding for TRP ion channels included in the study (13 genes, Table 1), only three genes encode for the cold-sensitive TRP ion channels: TRPA1, TRPC5, and TRPM8 [29]. The NM group down-regulated TRPC5 mRNA expression in all tissues (p < 0.05). As previously stated, all treatments down-regulated TRPC5 mRNA expression in the proximal uterus (p < 0.05). Besides, NM down-regulated TRPA1 mRNA expression in the uterus and isthmus (p < 0.05), and down-regulated TRPM8 mRNA expression in the isthmus (p < 0.05). Additionally, Semen-AI down-regulated TRPA1 mRNA expression in the distal uterus (p < 0.05). Three genes encoding for TRP ion channels are involved in the induction of CIPs: TRPM8, TRPV3, and TRPV4 [16,25]. No statistical differences were found for TRPV3 and TRPV4 mRNA expression. 2.3. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Analyses To categorize the function of the 39 DEGs detected, genes were mapped to terms in Gene Ontology (GO) database [35] (Figure 3). Out of the two main categories of the GO classification, the “biological process” was dominant. The predominant molecular function in the sperm-containing treatments was binding activity (GO:0005488) in both up- and down-regulated genes (p < 0.01). The predominant biological processes of the down-regulated genes in the sperm-containing treatments were biological regulation (GO:0065007) and cellular process (GO:0009987) followed by metabolic process (GO:0008152) (p < 0.01). The predominant biological process in the genes up-regulated by NM was cellular process (GO:0009987), followed by biological regulation (GO:0065007) (p < 0.05). No statistical differences were found within molecular functions and biological processes in the sperm-free SP treatments (p > 0.05). Molecular functions only differed in tissues where genes were up-regulated by the NM treatment (Figure 3a) predominantly in the infundibulum compared to endocervix (p < 0.05). The biological processes of the genes up-regulated by NM (Figure 3a) were mostly in the infundibulum and utero-tubal junction compared to the rest of the tissues (p < 0.05), whereas the biological processes of the genes down-regulated by NM (Figure 3a) were dominant in the ampulla and infundibulum compared to the endocervix and isthmus (p < 0.05). Similarly, the biological processes of the genes up-regulated by Semen-AI (Figure 3b) were dominant in the utero-tubal junction compared to the rest of the tissues (p < 0.001), whereas the biological processes of the genes down-regulated by Semen-AI (Figure 3b) dominated in the isthmus, ampulla, and infundibulum compared to the distal uterus (p < 0.05). The biological processes of the SP-AI group (Figure 3c) only differed among up-regulated genes, these being dominant in the distal uterus and utero-tubal junction compared to the endocervix, proximal uterus, isthmus, and ampulla (p < 0.05). Conversely, the biological processes of the genes up-regulated by SP-TOTAL (Figure 3d) appeared mostly in the infundibulum compared to the endocervix, distal uterus, utero-tubal junction, and ampulla (p < 0.05), whereas the genes down-regulated by SP-TOTAL (Figure 3d) dominated the proximal uterus compared to the rest of the tissues (p < 0.01). Altered transcripts were mapped to in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [36] and were associated with GO biological process subgroups (Figure 4). Results showed that the predominant GO biological processes were response to temperature stimulus and RNA transport followed by transmembrane transport, whereas the predominant KEGG pathway was inflammatory mediator regulation of TRP channels followed by mineral absorption. Furthermore, genes were classified by functionally grouped terms and pathways (Figure 5). The enrichment p-values were predominant in establishment of RNA localization and response to temperature stimulus followed by response to cold, RNA stabilization, and ribonucleoprotein complex assembly. Int. Int. J. Mol. Sci. 2020, 21, x 5333 109 of 21 23 Natural mating Semen-AI Up-regulated DEGs Up-regulated DEGs a b Down-regulated DEGs Down-regulated DEGs ng n n ity ss g n ity s n n n ity ity ss s s s s s es es es io io es es in io io io es tio iv tiv ce tiv ce iv di at ct nd at at ct oc oc oc oc oc oc la ct ct n ac ac ro ro du du ul iz z gu Bi Bi ra ra pr pr pr pr pr i pr l l eg rp rp ca ca ro ro ic ic re te te ic sm e ic sm e yt lr yt ep ep la la Lo Lo iv iv or ol ol or al al al ica lu lu ct ct ni ni R R ab ab p sp ic at at el el du du ga ns ga og og et et an C C C C ro a ro or or M M ol ol Tr Tr ep ep ti- ti- Bi Bi ul ul R R M M SP-AI Up-regulated DEGs SP-TOTAL Up-regulated DEGs c d Down-regulated DEGs Down-regulated DEGs g n n g n n ity ss ity ss n n ity s ity s s s s s es es es es es es in io io in io io io io iv tiv iv tiv ce ce nd at ct nd at ct at at oc oc oc oc oc oc ct ct ac ac ro ro du du ul iz ul iz Bi Bi ra ra pr pr pr pr pr pr l l eg eg rp rp ca ca ro ro ic ic rte rte ic ic sm e sm e yt yt lr lr ep ep la la Lo Lo iv iv ol ol po po al al a lu a lu ct ct ni ni R R ab ab ic ic at at el el du du ga ga ns ns g g et et C C C C lo lo ro ro a a or or M M o o Tr Tr ep ep ti- ti- Bi Bi ul ul R R M M Figure 3. Heatmap composition of Gene Ontology (GO) classification. Categories of the differentially expressed genes (p < Figure 3. Heatmap composition of Gene Ontology 0.05), up-regulated (brown) and (GO) classification. Categories down-regulated (blue), basedof the on differentially GO function expressed along genes (p