International Journal of Molecular Sciences Article Natural Mating Differentially Triggers Expression of Glucocorticoid Receptor (NR3C1)-Related Genes in the Preovulatory Porcine Female Reproductive Tract Mateo Ruiz-Conca 1,2 , Jaume Gardela 1,2 , Cristina Alicia Martínez 1 , Dominic Wright 3 , Manel López-Bejar 2,4 , Heriberto Rodríguez-Martínez 1 and Manuel Álvarez-Rodríguez 1,2, * 1 Department Biomedical and Clinical Sciences (BKV), BKH/OG, Linköping University, 58185 Linköping, Sweden; mateo.ruiz@uab.cat (M.R.-C.); jaume.gardela@uab.cat (J.G.); 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 Cerdanyola del Vallès, Spain; manel.lopez.bejar@uab.cat 3 Department of Physics, Chemistry and Biology, Faculty of Science and Engineering; Linköping University, 58183 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)72942-7883  Received: 13 June 2020; Accepted: 20 June 2020; Published: 22 June 2020  Abstract: Mating initiates dynamic modifications of gene transcription in the female reproductive tract, preparing the female for fertilization and pregnancy. Glucocorticoid signaling is essential for the homeostasis of mammalian physiological functions. This complex glucocorticoid regulation is mediated through the glucocorticoid receptor, also known as nuclear receptor subfamily 3 group C member 1 (NR3C1/GR) and related genes, like 11β-hydroxysteroid dehydrogenases (HSD11Bs) and the FK506-binding immunophilins, FKBP5 and FKBP4. This study tested the transcriptome changes in NR3C1/GR regulation in response to natural mating and/or cervical deposition of the sperm-peak ejaculate fraction collected using the gloved-hand method (semen or only its seminal plasma), in the preovulatory pig reproductive tract (cervix to infundibulum, 24 h after mating/insemination/infusion treatments). Porcine cDNA microarrays revealed 22 NR3C1-related transcripts, and changes in gene expression were triggered by all treatments, with natural mating showing the largest differences, including NR3C1, FKBP5, FKBP4, hydroxysteroid 11-beta dehydrogenase 1 and 2 (HSD11B1, HSD11B2), and the signal transducer and activator of transcription 5A (STAT5A). Our data suggest that natural mating induces expression changes that might promote a reduction of the cortisol action in the oviductal sperm reservoir. Together with the STAT-mediated downregulation of cytokine immune actions, this reduction may prevent harmful effects by promoting tolerance towards the spermatozoa stored in the oviduct and perhaps elicit spermatozoa activation and detachment after ovulation. Keywords: transcriptomics; microarrays; spermatozoa; mating; glucocorticoid; FKBP5; FKBP4; NR3C1; female reproductive tract; pig 1. Introduction Glucocorticoids (GCs) are steroid hormones essential for adaptation to stress, behavior, and reproduction. GC release is under circadian/ultradian pulsatile control of the hypothalamic–pituitary–adrenal (HPA) axis and its regulation is absolutely necessary for animal homeostasis. Undeniable new evidence suggests a crucial role of GCs during different reproductive steps, where they display central and peripheral regulation [1]. The balance between high and Int. J. Mol. Sci. 2020, 21, 4437; doi:10.3390/ijms21124437 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4437 2 of 17 low levels of GCs determines whether their biological activity mediates the correct functions or causes pathology. In this sense, it has long been assumed that GCs act negatively on reproductive function due to their role in chronic stress physiology [2]. However, while high chronic levels of the hormones in response to stress are pathological and affect fertility, basal levels of GCs are also essential for normal reproduction [1]. For instance, GCs undergo a significant rise prior to ovulation in most mammalian species, and although their increase may lead to impairment of their reproductive function [3], their presence is essential during embryonic/fetal development, parturition, and lactation [1,4,5]. GCs are also relevant for male fertility, and while GC-exposure drives the inhibition of steroidogenesis in the testis and apoptosis of germ cells [6], its deficiency impairs testicular function [5,7]. Although many studies address their influence in animal reproduction [8–11], the mechanisms by which GCs participate in energetically-demanding processes [12] (including sperm transport and storage in the female reproductive tract, the modulation of the immune response, and also their involvement in gene transcription signaling) are still not well known. Reproductive events that are initiated by either natural mating or artificial insemination (AI), such as sperm transport, sperm storage, fertilization, and the cascade of embryonic/fetal processes associated with implantation, placentation, cervical ripening, and final delivery, are all accompanied by inflammatory, immunological, and transcriptional responses in the reproductive tract. These events are often modulated by GCs, whose regulatory signaling action seem to be mediated by one particular receptor, the glucocorticoid receptor. This receptor, also known as the nuclear receptor subfamily 3 group C member 1 (NR3C1/GR), binds to the GC forming a complex that is translocated to the nucleus of the cells to modify gene transcription [13]. This translocation is done with the aid of partner molecules [10,14,15], including the peptidyl-prolyl cis/trans isomerase FK506-binding proteins (FKBP family proteins), which may regulate this translocation [16]. Thus, cortisol–glucocorticoid receptor complexes are responsible for the activation or repression of transcription of target genes (up to 10–20% of the whole genome in humans) [17,18] triggering, in turn, cascades with pleiotropic implications, that include reproductive, immune, and transgenerational effects [8,19]. In this regard, natural mating shapes reproductive physiology in a variety of different species due to a combination of the sensorial stimulation produced by penile buffeting, the presence of spermatozoa and/or the contact with seminal plasma (SP) [20–22]. The aforementioned mating factors seem to change gene and protein expression post-coitus, consequently modulating uterine and oviductal functions [23]. Such modifications elicited in the reproductive tract help to create a suitable environment necessary for sperm storage [24], gamete transport, pre-implantation development, increased angiogenesis, and also changes in the immune system pattern [25,26], including the attainment of a status of tolerance to foreign proteins and cells. Although GC signaling is essential for the establishment and maintenance of fertility [1], the up-stream triggers causing the modifications of NR3C1-related genes after natural mating or insemination in the preovulatory phase are yet to be determined. Therefore, the present study tested the hypothesis that (i) natural mating, (ii) the cervical deposition via AI of the sperm-peak ejaculate fraction, and (iii) AI of the sperm-free SP, all equally affect the expression of 22 genes involved in NR3C1/GR regulation in the preovulatory pig reproductive tract, 24 h after treatment. These treatments were performed on samples of mature fertile boars (n = 5) collected using the gloved-hand method. 2. Results 2.1. Gene Ontology of the Genes Related to the Glucocorticoid Receptor NR3C1 The 22 NR3C1-related genes of interest, directly or indirectly engaged in glucocorticoid receptor NR3C1 action and being potentially involved in reproductive functions and signaling (Figure 1), were analyzed. Besides NR3C1, these genes included the following: hydroxysteroid 11-beta dehydrogenase 1 and 2 (HSD11B1 and HSD11B2), the FK506-binding prolyl isomerase 5 and 4 (FKBP5 and FKBP4), the prostaglandin-endoperoxide synthase 1 and 2 (PTGS1 and PTGS2), the phospholipase Int. J. Mol. Sci. 2020, 21, 4437 3 of 17 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 17 A2 group IVB (PLA2G4B), the insulin-like growth factor binding protein 1 (IGFBP1), the heat shock protein the heatfamily shock A (HSP70) protein member family 8 (HSPA8), A (HSP70) memberthe8 signal transducer (HSPA8), the signaland activatorand transducer of transcription activator of 1, 2, 3,transcription 5A, 5B, and1,62,(STAT1, 3, 5A, 5B, and 6 STAT3, STAT2, (STAT1,STAT5A, STAT2, STAT3, STAT5B, STAT5A, STAT5B, and STAT6), and the STAT6), tumor the tumor protein P53 (TP53), protein the P53 (TP53), mediator complex thesubunit mediator complex 1 and subunit 14 (MED1 and1 MED14), and 14 (MED1 and the heat MED14), shock thefamily protein heat shock A (HSP70) protein family member A (HSP70) member 4 and member-4-like (HSPA44 and andmember-4-like HSPA4L), and(HSPA4 and protein heat shock HSPA4L), 90and alphaheat shockclass B family protein 90 member alpha family class B member 1 (HSP90AB1). 1 (HSP90AB1). Figure Figure 1. Schematicrepresentation 1. Schematic representation of of altered altered transcripts transcripts of interest of interest among among all tissues all tissues and treatments. and treatments. The The analysis of over-represented functional categories was performed using the Cytoscape v3.0.0 v3.0.0 analysis of over-represented functional categories was performed using the Cytoscape application ClueGo v2.0.3. application ClueGo v2.0.3.TheThe following following databases databases were were used:used: GeneGene Ontology Ontology (GO) (GO) subgroups subgroups biological processwhich biological process whichisisshown shown asas circles. circles. Terms Terms are are functionally functionally grouped grouped basedbased on shared on shared genes genes (kappa (kappascore) score)and andare shown are shownin different in different colors. TheThe colors. sizesize of the of nodes indicates the nodes the degree indicates of significance, the degree of where the biggest significance, where nodes correspond the biggest to the highest nodes correspond to thesignificance. The mostThe highest significance. significant term defines most significant term the defines name of the the name group.of the Thegroup. The following following ClueGo ClueGo parameters parameters were used: were used: biological biological process process database (BP; date: 19.05.2020); GO tree levels, 2–4 (first level = 0); minimum number of genes, 1; minimum database (BP; date: 19.05.2020); GO tree levels, 2–4 (first level = 0); minimum number of genes, 1; minimum percentage of genes, 1.2; GO term fusion; GO term connection restriction percentage of genes, 1.2; GO term fusion; GO term connection restriction (kappa score), 0.4; GO term (kappa score), 0.4; GO term initial grouping, grouping, groupinitial size group of 2 and size 50% of for 2 and group 50% for group merge. merge. The The resulting resulting network network was representation representation was manually rearranged after removing unnecessary manually rearranged after removing unnecessary terms. HSD11B1 and HSD11B2 (hydroxysteroid terms. HSD11B1 and HSD11B2 (hydroxysteroid 11-beta 11-beta 1 dehydrogenase dehydrogenase and 2), HSPA4, 1 and HSPA8 2), HSPA4, HSPA8 and(heat and HSP90AB1 HSP90AB1 (heat shock shock proteins 4, 8proteins and 90 alpha 4, 8 and 90 alpha family class B member 1), FKBP5 and FKBP4 (FK506-binding prolyl isomerase 5 and family class B member 1), FKBP5 and FKBP4 (FK506-binding prolyl isomerase 5 and 4), MED1 (mediator 4), MED1 (mediator complex subunit 1), NR3C1 (nuclear receptor subfamily 3 group C member 1), complex subunit 1), NR3C1 (nuclear receptor subfamily 3 group C member 1), PTGS1 and PTGS2 PTGS1 and PTGS2 (prostaglandin-endoperoxide synthase 1 and 2), STAT1, STAT2, STAT3, STAT5A, (prostaglandin-endoperoxide synthase 1 and 2), STAT1, STAT2, STAT3, STAT5A, STAT5B and STAT6 STAT5B and STAT6 (signal transducer and activator of transcription 1, 2, 3, 5A, 5B, and 6), TP53 (signal transducer and activator of transcription 1, 2, 3, 5A, 5B, and 6), TP53 (tumor protein 53). (tumor protein 53). Int. J. Mol. Sci. 2020, 21, 4437 4 of 17 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 17 2.2. Natural Mating and AI of Semen Components 2.2. Components Altered Altered the the Expression Expression of of Genes Genes Related Related to to the the Glucocorticoid Glucocorticoid Receptor Receptor NR3C1 NR3C1. Figure Figure 22 shows shows the the genes genes related related to to the the glucocorticoid glucocorticoid hormone hormone receptor receptor NR3C1 NR3C1 thatthat were were differentially differentially expressed (p (p <<0.05) 0.05)ininthethe proximal proximal uterus uterus (ProxUt), (ProxUt), utero-tubal utero-tubal junction junction (UTJ), (UTJ), and and isthmus (Isth) of the sow reproductive tract 24 h after (i) natural mating, isthmus (Isth) of the sow reproductive tract 24 h after i) natural mating, ii) cervical insemination with(ii) cervical insemination withofthe the first portion first portion sperm-rich of sperm-rich fraction fraction (semen-AI), (semen-AI), or iii) or (iii) cervical cervical infusion with theinfusion with sperm-free the sperm-free seminal plasma of this portion (SP-AI). Interestingly, seminal plasma of this portion (SP-AI). Interestingly, some of the genes were some of the genes were similarly downregulated (HSD11B1,FKBP4, downregulated(HSD11B1, FKBP4, PTGS2, PTGS2, STAT5A) andand STAT5A)or upregulated (FKBP5) or upregulated in these (FKBP5) inthree thesegenital three compartments, considered genital compartments, the most considered thebiologically relevantrelevant most biologically during the preovulatory during phase [27,28]. the preovulatory phase The overall [27,28]. Thedifferentially-expressed genes, fromgenes, overall differentially-expressed cervixfrom (Cvx)cervix to infundibulum (Inf) are represented (Cvx) to infundibulum in (Inf) are Supplementary Figure S1. represented in Supplementary Figure S1. Figure Figure 2.2. Differentially-expressed genes (DEGs) in proximalproximal uterus uterus (ProxUt), (ProxUt), utero-tubal utero-tubal junction junction (UTJ), (UTJ), and and isthmus isthmus (Isth) (Isth) tissues, tissues, ordered ordered by treatments treatments (natural mating, semen-artificial semen-artificial insemination insemination (AI), (AI), or or sperm-free sperm-free (SP)-AI). (SP)-AI). Upregulated Upregulatedgenes genes(p(p<< 0.05) are marked marked in in green, green, while while downregulated downregulated genes (p < genes (p < 0.05) are shown in red. Color grading is displayed, ranging from 1(upregulated) 0.05) are shown in red. Color grading is displayed, ranging from 1 (upregulated)to to−1 -1 (downregulated), (downregulated), separately for for every every treatment. treatment. False Falsediscovery discoveryrates rates(q (q < 0.05) < 0.05) areare noted noted with with an an asterisk. asterisk. Natural Natural mating mating (Figure (Figure 3) 3) was, was, byby far, far, the the treatment treatment that that caused caused the the largest largest changes changes to to gene gene expression. expression. In mating, 23 differential expression changes were registered (10 in ProxUt, five in In mating, 23 differential expression changes were registered (10 in ProxUt, five in UTJ, UTJ, and and eight eightinin Isth), withwith Isth), 17 out 17ofoutthe of 23 affected genes being the 23 affected downregulated genes (73.9%) in the being downregulated reproductive (73.9%) in the tract reproductive (p 70% sperm motility and with >75% morphologically-normal spermatozoa. The ejaculate sperm-rich first portion [89] was used for the AI infusion in the semen-AI or SP-AI groups. Seminal plasma (SP-AI) was harvested through double centrifugation (1500 × g/ 10 min) and checked as sperm- and somatic cell-free. All sows were subjected to surgery (mid-laparotomy) under general anesthesia [89] to remove samples from the cervix, uterus, and oviduct. All samples were plunged into liquid nitrogen and later stored (−80 ◦ C) until analyzed. Ovaries contained only un-ovulated follicles, in similar numbers among groups (22.30 ± 7.29, mean ± SD) and the condition of pre-ovulation was confirmed by the ratio of circulating estrogen:progesterone in blood plasma [89]. 4.5. Microarray Hybridization and Scanning Total RNA was extracted from tissue segments with Trizol using the protocols described elsewhere [27]. Complementary DNA (cDNA) of each sample was obtained from RNA (250 ng) of each sample by using the GeneChip®Whole Transcript Plus reagent kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer protocol. The hybridization of cDNA mixture of each sample was made using labeled single-strand fragments of cDNA (3.5 µg; 41 µL) and hybridization master mix (109 µL). This cocktail was incubated at 99 ◦ C for 5 min, decreasing the temperature to 45◦ C afterwards. The cocktail hybridization mix (130 µL) of each sample was then loaded on a microarray chip specific for porcine species (GeneChip®Porcine Gene 1.0 ST Array, ThermoFisher Scientific, Sweden) for incubation under rotation (60 revolutions per minute) at 45 ◦ C for 16 h. After being Int. J. Mol. Sci. 2020, 21, 4437 12 of 17 incubated, the hybridized array was unloaded, washed, and stained (GeneChip®Fluidics Station 450, Affymetrix). The array chip was then scanned by using GeneChip®scanner GCS3000. 4.6. Microarray Data Analysis All data obtained from each array was analyzed with the Transcriptome Analysis Console (TAC; version 4.0) from Affymetrix. Briefly, robust multi-array average (RMA) normalization, computing average intensity values by background adjustment, quantile normalization among arrays, and logarithmic transformation was performed in order to obtain the values of expression of each of the transcripts. Only genes related to the glucocorticoid receptor (NR3C1/GR) were assessed in detail. These 22 genes were reported as related to NR3C1 by using a combination of the internet-based tools. These interaction networks included protein and genetic interactions, pathways, and co-expression. Also, PANTHER (Protein Analysis Through Evolutionary Relationships) classification system for gene ontology (GO) [31] of biological process and molecular function was used for the analysis of the functions of the analyzed genes. Graphical illustration of overrepresented GO terms was produced with the Cytoscape v3.0.0 application CluePedia v2.0.3 [91]. Statistics of the normalized gene expression were determined using a linear model with an empirical Bayes method implemented in the specific package of Linear Models for Microarray Analysis (“Limma”). The statistical analyses were performed to detect differential expression of transcripts using a Benjamini−Hochberg false discovery rate (FDR) (q < 0.05) and a principal component analysis (PCA)-based p-value correction for type-I errors made with a statistical cut off p < 0.05 or FDR q < 0.05) [29], which was completed using ClustVis (BETA) web tool [92]. The principal component analysis clustered, after linear transformation, multivariate data ordered based on the variance. The prediction ellipses showed that the probability for a new observation from each group would be inside the ellipse (0.05 error). All the redundant or uncharacterized transcripts were excluded to obtain a final database of differentially-expressed genes. The list of genes found to be differentially expressed were then searched for functional pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [30]. 5. Conclusions Overall, the results of this study indicate that natural mating seems to produce a differential response in the reproductive tract of the sow, compared to the use of AI, at least in the preovulatory phase. In porcine, it seems clear that in terms of effects on gene expression, some inherent effects of natural mating on GC regulation genes, could not be mimicked by AI. Moreover, the genes involved in the glucocorticoid receptor control (FKBP4 and FKBP5), cortisol availability (HSD11B1 and HSD11B2), and JAK-STAT signaling (STAT5A), exert a collective glucocorticoid-avoiding response that may prevent detrimental effects in the sperm reservoir and help sperm activation and detachment close to the time of ovulation. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/12/ 4437/s1. Figure S1. Differentially expressed genes (DEGs) of cervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp) and infundibulum (Inf), ordered by treatments (natural mating, Semen-AI or SP-AI). Numbers represent the fold change for each gene on each tissue, compared to control. Upregulated genes (p < 0.05) are marked in green, while downregulated genes (p < 0.05) are shown in red colour. Colour grading is displayed ranging from 1 (upregulated), and from -1 (downregulated) in every treatment, separately. FDRs (q < 0.05) is noted in bold. Figure S2. Principal Component Analysis (PCA) of (A) proximal uterus (ProxUt), (B) uterotubal junction (UTJ) and (C) isthmus (Isth) was depicted. The prediction ellipses show the probability for a new observation from each group will be inside the ellipse (0.05 error). Principal component 1 (PC1) and principal component 2 (PC2) explain percentage of the total variance, respectively. Figure S3. Heat map of (A) proximal uterus (ProxUt), (B) utero-tubal junction (UTJ) and (C) isthmus (Isth) was depicted. Data was clustered using correlation distance and average linkage. Table S1. Kyoto Encyclopedia of Gens and Genomes (KEGG) Pathways of genes differentially expressed by natural mating. Upregulation is represented in bold and downregulation in non-bold (p < 0.05) in each tissue: cervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp) and infundibulum (Inf). Also, false discovery rates (FDRs) are shown in red (q < 0.05). Table S2. KEGG Pathways of genes differentially expressed by cervical insemination of the first portion of the sperm-rich ejaculate fraction (Semen-AI). Upregulation is shown in bold and downregulation in non-bold (p < 0.05) in each tissue: cervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), Int. J. Mol. Sci. 2020, 21, 4437 13 of 17 utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp) and infundibulum (Inf). Table S3. KEGG Pathways of genes differentially expressed by cervical insemination of the sperm-free seminal plasma of the first portion of the sperm-rich fraction (SP-AI). Upregulation is shown in bold and downregulation in non-bold (p < 0.05) in each tissue: cervix (Cvx), distal uterus (DistUt), proximal uterus (ProxUt), utero-tubal junction (UTJ), isthmus (Isth), ampulla (Amp) and infundibulum (Inf). Author Contributions: Conceptualization, M.Á.-R., H.R.-M., and D.W.; methodology, M.R.-C., C.A.M., M.Á.-R., and H.R.-M.; software, M.R.-C., C.A.M., and M.A.R.; validation, M.R.-C. and M.Á.-R.; data curation, M.R.-C. and M.Á.-R.; writing—original draft preparation, M.R.-C.; writing—review and editing, J.G., M.A.R., H.R.-M., D.W., and M.L.-B.; visualization, M.R.-C.; supervision, M.A.R., H.R.-M., and M.L.-B.; project administration, M.A.R. and H.R.-M.; funding acquisition, H.R.-M. and M.Á.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Research Council FORMAS, Stockholm (Project 2017-00946 and Project 2019-00288), the Swedish Research Council (Vetenskapsrådet, VR; project 2015-05919), the Government of Spain—Ministry of Education, Culture and Sports (Training program for Academic Staff FPU15/06029), the Seneca Foundation (20780/PD/18) Murcia (Spain), and the Government of Catalonia-AGAUR, co-financed with the European Social Fund (2018 FI_B 00236). Acknowledgments: Annette Molbaek and Åsa Schippert, for LiU core facility, for expert assistance when running the microarrays. To Linköping University for supporting the open access funding. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Whirledge, S.; Cidlowski, J.A. 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