animals Article Semen Modulates Inflammation and Angiogenesis in the Reproductive Tract of Female Rabbits Jaume Gardela 1,2 , Amaia Jauregi-Miguel 3 , Cristina A. Martinez 1 , Heriberto Rodríguez-Martinez 1 , Manel López-Béjar 2,4 and Manuel Álvarez-Rodríguez 1,2, * 1 Department of Biomedical and Clinical Sciences (BKV), Division of Children’s and Women Health (BKH), Obstetrics and Gynecology, Linköping University, 58185 Linköping, Sweden; 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 Bellaterra, Spain; manel.Lopez.Bejar@uab.cat 3 Division of Molecular Medicine and Virology (MMV), Linköping University, 58185 Linköping, Sweden; amaya.jauregi.miguel@liu.se 4 College of Veterinary Medicine, Western University of Health Sciences, Pomona, CA 91766, USA * Correspondence: manuel.alvarez-rodriguez@liu.se  Received: 19 October 2020; Accepted: 18 November 2020; Published: 25 November 2020  Simple Summary: In mammals, the expression of regulatory genes is modified by the interaction between semen and the female reproductive tract. This study intends to unveil how mating or insemination with sperm-free seminal plasma, as well as the presence of preimplantation embryos, affects inflammation and angiogenesis in different segments of the reproductive tract of female rabbits. Gene expression of anti-inflammatory cytokines and angiogenesis mediators was analyzed in segmented tracts (cervix to infundibulum) in response to mating and sperm-free seminal plasma infusion. Moreover, the gene expression at different times post-mating was also analyzed. Results showed that gene expression changes were mainly localized in the uterus in the natural mating group, describing a clear temporal variation, while limited to the oviduct in the sperm-free seminal plasma group. These changes suggest an early response in the uterus and late modulation in the oviduct, distinctly demonstrating that semen and seminal plasma, through their interaction with the female reproductive tract, can differentially modulate the expression of anti-inflammatory and angiogenesis mediators. Abstract: The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGFβ1–3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (GnRH) stimulation (control), natural mating (NM) or vaginal infusion with sperm-free SP (SP-AI). Additionally, segmented samples were also obtained at 10, 24, 36, 68 or 72 h after GnRH-stimulation and natural mating (Experiment 2). The results of gene expression, analyzed by quantitative PCR, showed that NM effects were mainly localized in the uterine tissues, depicting clear temporal variation, while SP-AI effects were restricted to the oviduct. Changes in anti-inflammatory and angiogenesis mediators indicate an early response in the uterus and a late modulation in the oviduct either induced by semen or preimplantation embryos. This knowledge could be used in the implementation of physiological strategies in breeding systems to face the new challenges on rabbit productivity and sustainability. Animals 2020, 10, 2207; doi:10.3390/ani10122207 www.mdpi.com/journal/animals Animals 2020, 10, 2207 2 of 14 Keywords: gene expression; endometrium; oviduct; spermatozoa; seminal plasma; inflammation; angiogenesis; rabbit 1. Introduction Considered either laboratory animals, pets, invasive species or livestock, the rabbit (Oryctolagus cuniculus) is one of the most versatile and multipurpose animal species bred by humans [1–3]. Subjected today to intense production for meat and fur, does are affected by requirements on fertility and lifespan [4]. Contrary to other livestock species, rabbits require the generation of genital-somatosensory signals during mating to generate the preovulatory peak of gonadotropin-releasing hormone (GnRH) [5,6]. The consequent release of luteinizing hormone from the anterior pituitary induces ovulation [5]. Despite requiring hormonal stimulation if artificial insemination (AI) is used [7], this technique is extensively employed, giving similar or better results than natural mating [8] when using fresh or cooled semen [9]. The success of AI with diluted semen in several species suggest that seminal plasma (SP) components, excluding spermatozoa, are not required for pregnancy [10]. However, in some species, such as rodents or pigs, reproductive success and pregnancy can be jeopardized when the SP signaling is disrupted [10]. Several studies demonstrated that SP has multiple effects on the female reproductive tract essential for conceptus and pregnancy, improving reproductive performance [10]. In different species of mammals, the direct interaction between the female reproductive tract and semen, as well as the sensorial stimulation during the act of mating [11], induce molecular and cellular modifications into the female reproductive tract [12–14], modulating the immune system and optimizing the reproductive outcomes [15]. The immune response generated as a result of mating leads to a suitable environment for embryo survival, implantation success, optimal fetal and placental development and the overall reproductive process [16]. The first description of female post-mating inflammation responses was made by observing rabbits in 1952 [17]. The observations of McDonald and collaborators showed a leucocytic influx in the uterus in response to SP but not by sterile spermatozoa administration [17]. Later studies demonstrated that both SP and spermatozoa triggered a uterine leucocytic response in rabbits [18,19]. Contrary to the vagina, cervix and uterus, the oviduct seems to respond to insemination without a reduction of the spermatozoa population at the time of ovulation [20], providing the optimal conditions for spermatozoa survival until fertilization takes place [7]. The interaction between spermatozoa and oviductal epithelial cells could regulate the immunological environment of the oviduct-inducing anti-inflammatory cytokines [21], like transforming growth factor beta 1 (TGFβ1) or interleukin-10 (IL10), protecting spermatozoa from immune responses [13]. The transforming growth factor beta (TGFβ) family regulates many cellular activities, including cell growth, proliferation, differentiation, tissue remodeling, extracellular matrix formation, control of cell surface molecules, immunoregulation, angiogenesis and apoptosis [22]. The TGFβ belong to a large superfamily of cytokines composed of three 25 kDa homodimeric proteins (TGFβ1–3) whose biological effects are mediated by three transforming growth factor beta receptors (TGFβR1–3) [22,23]. The IL10, first described as cytokine synthesis inhibitory factor or CSIF [24], is a cytokine that inhibits inflammatory responses preventing the secretion of inflammatory cytokines and regulating the differentiation and proliferation of immune cells [25]. The vascular endothelial growth factor (VEGF, also referred to as VEGF-A), a heparin-binding homodimeric glycoprotein, is a mitogen for endothelial cells and a potent inducer of angiogenesis [26] that also promotes vascular permeability [27]. VEGF protein family includes other factors like VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (P1GF) [28,29]. VEGF-A is expressed in the endometrium of several species [30–32], being proposed in the rabbit as a local signal between the implantation embryo and vascular structures in the receptive endometrium [33]. Animals 2020, 10, 2207 3 of 14 This study aimed to determine whether natural mating and/or sperm-free SP infusion modulates the expression of anti-inflammatory cytokines and angiogenesis mediator genes (IL10, TGFβ1–3 and VEGF-A) in the maternal environment of the doe at 20 h post-exposure. Moreover, we determine the Animals 2020, 10, x 3 of 14 expression of these mediators when early embryo development occurs along the reproductive tract of the doe up to in VEGF-A) 72theh post-mating. Additionally, maternal environment of thewe doeanalyzed the spatial changes at 20 h post-exposure. of these Moreover, genes along we determine the the tubular reproductive expression of thesetract of the when mediators doe. early embryo development occurs along the reproductive tract of the doe up to 72 h post-mating. Additionally, we analyzed the spatial changes of these genes along 2. Materials and the tubular Methods tract of the doe. reproductive 2.1. Ethics Statement 2. Materials and Methods Rabbits were handled according to the Spanish Royal Decree 1201/2005 (BOE, 2005: 252:34367-91) 2.1. Ethics Statement and the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on Rabbits the protection of were handled animals usedaccording to thepurposes for scientific Spanish Royal (2010;Decree 1201/2005 276:33–79). The(BOE, 2005: 252:34367- Committee of Ethics and 91) and the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 Animal Welfare of the Universitat Autónoma de Barcelona, Spain, approved this study (Expedient #517). on the protection of animals used for scientific purposes (2010; 276:33–79). The Committee of Ethics and Animal Welfare of the Universitat Autónoma de Barcelona, Spain, approved this study 2.2. Animals and Experimental Design (Expedient #517). Animals were housed in the nucleus colony at the farm of the Institut de Recerca i Tecnologia 2.2. Animals and(IRTA-Torre Agroalimentaries Experimental Marimon, Design Caldes de Montbui, Barcelona, Spain) under a controlled photoperiod and were Animals temperature housed in[34]. Single cage the nucleus colony equipped at the farm with plastic of the footrests, Institut a feeder de Recerca (restricted i Tecnologia to 180 g/day of an all-mash Agroalimentaries pellet) (IRTA-Torre and nipple-drinkers Marimon, Caldes de Montbui, (ad libitum access Barcelona, Spain)tounder water) were used for a controlled each photoperiod rabbit. and temperature [34]. Single cage equipped with plastic footrests, a feeder (restricted to 180 g/day of Six New Zealand an all-mash White pellet) (NZW) andadult nipple-drinkers bucks (from (ad seven libitumtoaccess to water) thirteen monthswere used old) for each were included rabbit. in the study. At 4.5 months of age, the bucks were started to be trained with a homemade polyvinyl Six New Zealand White (NZW) adult bucks (from seven to thirteen months old) were included chloride artificial vagina at 50 ◦ C to ensure 41–42 ◦ C at the time of semen collection. One ejaculate in the study. At 4.5 months of age, the bucks were started to be trained with a homemade polyvinyl was collected per male, discarding ejaculates that contained urine and calcium carbonate deposits on chloride artificial vagina at 50 °C to ensure 41–42 °C at the time of semen collection. One ejaculate visualwas inspection. collected per male, discarding ejaculates that contained urine and calcium carbonate deposits on Two visualseparate experiments were performed in this study (Figure 1). In Experiment 1, 9 NZW adult inspection. does wereTwo randomly separateallocated experiments intowere three experimental performed in thisgroups. Sequential study (Figure 1). In segments Experimentof1,the right side 9 NZW of female adult reproductive does were randomlytractsallocated were retrieved into threeafter 20 h post-induction experimental of the segments groups. Sequential ovulation of with 0.03 mg the right GnRH side (Fertagyl ® ,reproductive of female tracts were Esteve Veterinaria, retrievedSpain) Barcelona, after 20intramuscularly h post-induction(im)of the ovulation (control withovulation; of the 0.03 = 3), 20 mg nGnRH (Fertagyl ®, Esteve Veterinaria, Barcelona, Spain) intramuscularly (im) (control of the control, h post-induction of the ovulation with 0.03 mg GnRH im and sperm-free SP vaginal ovulation; control, n = 3), 20 h post-induction infusion (SP-AI, n = 3) and 20 h post-induction of of thethe ovulation withwith ovulation 0.03 mg 0.03GnRH im and im mg GnRH sperm-free and natural SP vaginal infusion (SP-AI, n = 3) and 20 h post-induction of the ovulation with 0.03 mg GnRH im mating (NM, n = 3). In Experiment 2, 15 NZW adult does were sequentially euthanized at 10, 24, 36, 68 and natural mating (NM, n = 3). In Experiment 2, 15 NZW adult does were sequentially euthanized or 72 h post-induction of the ovulation with 0.03 mg GnRH im and natural mating (n = 3/collection at 10, 24, 36, 68 or 72 h post-induction of the ovulation with 0.03 mg GnRH im and natural mating (n time).= 3/collection The 10 h group was10established time). The h group was as the reference established group togroup as the reference compare the ovulatory to compare the ovulatorymoment (10 h moment post-mating) with some different time-point steps of the preimplantation (10 h post-mating) with some different time-point steps of the preimplantation embryo embryo development: 2–4 cell embryo (24 development: h),cell 2–4 8-cell embryo embryo (36 (24 h), h), early 8-cell embryo morula (36 h), (68 h)morula early and morula (72 morula (68 h) and h). (72 h). Figure 1. Representation Figure of theofexperimental 1. Representation design the experimental and tissue design sections and tissue obtained sections from does. obtained from Sequential does. Sequential tissue segments derived from does were endocervix, distal uterus, proximal uterus, tissue segments derived from does were endocervix, distal uterus, proximal uterus, utero-tubal junction, utero- distaltubal junction, isthmus, distaland ampulla isthmus, ampulla Intramuscular infundibulum. and infundibulum. Intramuscular injection of 0.03 mginjection of 0.03 mg gonadotropin-releasing gonadotropin-releasing hormone (GnRH) was used to induce ovulation hormone (GnRH) was used to induce ovulation in all groups of both experiments. in all groups of both experiments. Animals 2020, 10, 2207 4 of 14 2.3. Mating and Semen Collection Two randomly selected bucks were used to mate the does included in the mating group of Experiment 1 and Experiment 2 after the hormonal induction of the ovulation with 0.03 mg GnRH im. Additionally, semen was collected from the same rabbit bucks through an artificial vagina, as described above. The sperm-free SP was obtained after centrifugation at 2000× g for 10 min and checked for the absence of spermatozoa. The harvested sperm-free SP was immediately used for sperm-free SP vaginal infusions of Experiment 1. 2.4. Tissue Sample Collection For each experimental condition, seven consecutive tissue sections from the female reproductive tracts (endocervix, distal uterus, proximal uterus, utero-tubal junction, ampulla, isthmus and infundibulum; Figure 1) were obtained after the euthanasia of the does [34]. The tissue segments were retrieved and stored in RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA) at −80 ◦ C. Before tissue segmentation of does included in experiment 2, the oviduct was isolated and flushed (phosphate-buffer saline supplemented with 5% fetal calf serum and 1% antibiotic-antimycotic solution) to collect the embryos, which were examined by number and developmental stage. The number of ovulated ovarian follicles (5.08 ± 2.06 follicles, mean ± standard deviation (SD)) and embryos (4.67 ± 3.14, mean ± SD) were counted for each side of the reproductive organs, as previously published elsewhere [34]. Briefly, at 24 h (2 and 4-cell), 36 h (8-cell stage), 68 h (early morula) and 72 h (morula), the embryos were collected by the method described above. 2.5. Quantitative PCR Analyses Briefly, total RNA was extracted from the tissue segments following a TRIzol-based protocol [34]. The RNA concentration of the extracts was determined from the absorbance of 260 nm with Thermo Scientific NanoDropTM 2000 (Fisher Scientific, Gothenburg, Sweden). The quality of the RNA was determined by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), using the samples with an RNA integrity number higher than 8. High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™, Foster City, CA, USA) was used to synthesize the first-strand cDNA for quantitative polymerase chain reaction (qPCR) analyses (CFX96™; Bio-Rad Laboratories, Inc; Hercules, CA, USA). Following our previous qPCR protocol [34], the gene relative expression levels were quantified using the Pfaffl method [35]. The primer sequences, product sizes and efficiencies are shown in Table 1. For the β-ACTIN gene, commercial gene-specific PCR primers for rabbit samples were used (PrimePCR™SYBR® Green Assay: ACTB, Rabbit; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Product sizes were confirmed by loading the amplicons in an agarose gel using a gel imaging system (ChemiDoc XRS+ System, BioRad Laboratories, Inc., Hercules, CA, USA). Table 1. Primers used for the quantitative PCR analyses. Gene Primer Sequence (50 –30 ) Product Size (bp) Efficiency (%) F: commercial, not available 120 88.6 β-ACTIN R: commercial, not available F: TTGTTAACCGAGTCCCTGCT 209 107.5 IL10 R: TTTTCACAGGGGAGAAATCG F: CACCATTCACAGCATGAACC 129 99.3 TGFβ1 R: TTCTCTGTGGAGCTGAAGCA F: CCGGAGGTGATCTCCATCTA 236 94.7 TGFβ2 R: AGGTACCCACAGAGCACCTG F: CACGAACCCAAGGGCTACTA 186 97.5 TGFβ3 R: GGTCCTCCCGACGTAGTACA F: GGAGACAATAAACCCCACGA 219 87.0 VEGF-A R: CTGCATGGTGACGTTGAACT β-ACTIN: β-actin, IL10: interleukin 10, TGFβ1: transforming growth factor β 1, TGFβ2: transforming growth factor β 2, TGFβ3: transforming growth factor β 3, VEGF-A: vascular endothelial growth factor A, F: forward, R: reverse, A: adenine, C: cytosine, G: guanine, T: thymine, bp: base pair. Animals 2020, 10, 2207 5 of 14 2.6. Statistical Analyses CFX Maestro™ 1.1 software version 4.1.2433.1219 (Bio-Rad Laboratories, Inc; Hercules, CA, USA) was used to export all data. Normal distribution and homoscedasticity of the data were analyzed using the Shapiro–Wilk normality test and Levene’s test. Non-normal data distribution was restored using Log(x) transformation prior to analysis. R version 3.6.1. [36] was used to conduct the statistical analyses with nlme [37] to perform linear mixed-effects (LME) models and multcomp [38] to perform pairwise comparisons adjusted by Tukey’s test. The threshold for significance was set at p < 0.05. Data are presented as median (minimum, maximum) unless otherwise stated. A first LME model included the treatments of Experiment 1 (control, SP-AI, NM) as fixed effects and the females as the random part of the model. Pairwise comparisons were adjusted by Tukey’s test. A second LME model included the different collection times of Experiment 2 (10, 24, 36, 68 and 72 h post-mating) as fixed effects and the females as the random part of the model. Post hoc comparisons were performed using Tukey’s multiple comparisons test. The differential expression changes in qPCR results among tissues in both Experiments were re-analyzed separately. The utero-tubal junction was used as an arbitrary anatomical compartment reference among all tissues examined to compare the gene expression changes per gene [34], issued both by control, NM or SP-AI (Experiment 1) or by different times post-mating (Experiment 2). The LME model was followed by Tukey’s multiple comparison test to analyze the differences among each anatomical region of the female reproductive tract. Data are presented as median (minimum, maximum) unless otherwise stated. Data on the differential expression among tissues are presented as Row Z-scores. 3. Results 3.1. Natural Mating and Sperm-Free Seminal Plasma Infusion Differentially Modulated Anti-Inflammatory Cytokines and VEGF-A Genes Differences in IL10, TGFβ1, TGFβ2, TGFβ3 and VEGF-A expression among groups included in Experiment 1 are displayed in Figure 2 (and Figure S1). The NM treatment downregulated the IL10 expression in the utero-tubal junction (p < 0.05); upregulated the TGFβ1 and TGFβ3 expression in the distal (p < 0.01) and proximal uterus (p < 0.001); upregulated the TGFβ1 and TGFβ2 expression in the infundibulum (p < 0.01); and upregulated the VEGF-A expression in the distal uterus (p < 0.05). The SP-AI treatment upregulated the TGFβ1 expression in the proximal uterus (p < 0.01), upregulated the TGFβ1 and TGFβ3 expression in the infundibulum (p < 0.01), and upregulated the VEFG-A expression in the infundibulum (p < 0.01). Animals 2020, 10, x 6 of 14 Animals 2020, 10, 2207 6 of 14 M C M C I I M C I -A -A -A h_ h_ h_ N N N SP SP SP h_ h_ h_ 20 20 20 h_ 20 h_ 20 h_ 20 20 20 20 TGF 3 - ProxUt 150 a 100 50 b b 0 M C I M C I -A -A h_ h_ N N SP SP h_ h_ 20 20 h_ h_ 20 20 20 20 M C I -A h_ N SP h_ 20 h_ 20 20 Figure 2. Results of Experiment 1. Gene expression of (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ3 and (e) VEGF-A in differentially expressed segments (p < 0.05) (DistUt: distal uterus, ProxUt: proximal uterus, Figure 2. Results of Experiment 1. Gene expression of (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ3 and UTJ: utero-tubal junction, Amp: ampulla and Inf: infundibulum) of the does internal reproductive (e) VEGF-A in differentially expressed segments (p < 0.05) (DistUt: distal uterus, ProxUt: proximal tract after 20 h of the induction of the ovulation with 0.03 mg gonadotropin-releasing hormone (GnRH) uterus, UTJ: utero-tubal junction, Amp: ampulla and Inf: infundibulum) of the does internal intramuscularly (20 h_C, n = 3), 20 h post-GnRH-stimulation and seminal plasma vaginal infusion reproductive tract after 20 h of the induction of the ovulation with 0.03 mg gonadotropin-releasing (20h_SP-AI, n = 3) and 20 h post-GnRH-stimulation and natural mating (20 h_NM, n = 3). Fold changes hormone (GnRH) intramuscularly (20 h_C, n = 3), 20 h post-GnRH-stimulation and seminal plasma relative to the control of the ovulation group are shown. Different letters (a,b) represent statistical vaginal infusion (20h_SP-AI, n = 3) and 20 h post-GnRH-stimulation and natural mating (20 h_NM, n differences between groups (p < 0.05). Median (minimum, maximum). = 3). Fold changes relative to the control of the ovulation group are shown. Different letters (a,b) representGene 3.2. Temporal statistical differences Expression between groups (pcytokines of Anti-Inflammatory < 0.05). Median (minimum, and VEGF-A at 10maximum). h up to 72 h Post-Mating Differences 3.2. Temporal Gene IL10, TGFβ1, inExpression TGFβ2, TGFβ3 and of Anti-Inflammatory VEGF-A cytokines andexpression VEGF-A atin10doesh upsampled at different to 72 h Post-Mating times post-mating are displayed in Figure 3 (and Figure S2), with 10 h post-mating as the control group. Differences The IL10 in IL10, expression was TGFβ1, TGFβ2, TGFβ3 downregulated and at 36, 68 VEGF-A and expression in 72 h post-mating in does sampled atand the endocervix different distal times post-mating are displayed in Figure 3 (and Figure S2), with 10 h post-mating uterus (p < 0.001); downregulated at 24, 36, 68 and 72 h post-mating in the utero-tubal junction as the control and group. The IL10 infundibulum (pexpression < 0.001); andwas downregulated upregulated at 68 at and36,7268hand 72 h post-mating post-mating in the in the distal endocervix isthmus and (p < 0.05). distal uterus (p < 0.001); downregulated at 24, 36, 68 and 72 h post-mating in the utero-tubal The TGFβ1 expression was downregulated at 36, 68 and 72 h post-mating in the distal uterus (p < 0.05); junction and and infundibulum downregulated(pat< 24, 0.001); andand 36, 68 upregulated at 68 and 72 h post-mating in72theh infundibulum post-mating in(pthe distal isthmus < 0.001). The TGFβ2(p < 0.05). The TGFβ1 expression was downregulated at 36, 68 and 72 h post-mating in the expression was downregulated at 24, 36, 68 and 72 h post-mating in the distal uterus and infundibulum distal uterus (p < 0.05); and downregulated at 24, 36, 68 and 72 h post-mating in the infundibulum (p < 0.001); downregulated at 24 h post-mating in the proximal uterus (p < 0.01); and downregulated at (p < 0.001). The TGFβ2 24 and 72expression was in h post-mating downregulated the utero-tubalat 24, 36,(p68< and junction 0.05).72The h post-mating in thewas TGFβ3 expression distal uterus and downregulated infundibulum (p < 0.001); downregulated at 24 h post-mating in the proximal uterus (p < 0.01); and Animals 2020, 10, x 7 of 14 Animals 2020, 10, 2207 7 of 14 downregulated at 24 and 72 h post-mating in the utero-tubal junction (p < 0.05). The TGFβ3 expression was downregulated at 36 h post-mating in the endocervix (p < 0.05); downregulated at 36, 68 and 72 at 36 h post-mating h post-mating in theindistal the endocervix isthmus (p(p < 0.05); < 0.05); anddownregulated downregulated at 36, 68 36, at 24, and6872and h post-mating in the 72 h post-mating distal isthmus (p < 0.05); and downregulated at 24, 36, 68 and 72 h post-mating in the in the infundibulum (p < 0.001). The VEGF-A expression was upregulated at 24 h post-mating in the infundibulum (p < 0.001). The utero-tubal VEGF-A junction (p < expression was upregulated 0.05); downregulated at 24 at 24 and 72hhpost-mating post-matingininthe theutero-tubal ampulla (pjunction < 0.05); (p < 0.05); downregulated at 24 and 72 h post-mating in the ampulla (p < 0.05); and downregulated at 24, 36, 68 and 72 h post-mating in the infundibulum (p < 0.001). and downregulated at 24, 36, 68 and 72 h post-mating in the infundibulum (p < 0.001). h h h h h 10 24 36 68 72 h h h h h h h h h h h h h h h h h h h h h h h h h 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 TGF 1 - UTJ TGF 1 - Amp 8 3 a,b a 6 2 4 a,b 1 2 b,c a,b,c c a,b a a,b b 0 0 h h h h h h h h h h h h h h h h h h h h 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 h h h h h h h h h h h h h h h h h h h h 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 TGF 3 - Cvx TGF 3 - UTJ 2.0 5 a a a 4 a 1.5 3 a 1.0 a,b 2 a,b a,b 0.5 1 b b 0.0 0 h h h h h h h h h h h h h h h h h h h h 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 h h h h h h h h h h h h h h h 10 24 36 68 72 10 24 36 68 72 10 24 36 68 72 Figure 3. Results of Experiment 2. Gene expression of (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ3 and Figure (e) 3. Results VEGF-A of Experiment was statistically 2. Gene in significant expression of (a) IL10, rabbit endocervix (b) TGFβ1, (Cvx), (c) TGFβ2, distal uterus (d) TGFβ3 (DistUt), and proximal (e) VEGF-A uterus was utero-tubal (ProxUt), statistically junction significant in rabbit (UTJ), distalendocervix (Cvx), isthmus (Isth), distal (Amp) ampulla uterus and (DistUt), proximal infundibulum uterus (Inf) (ProxUt), after 10, 24, utero-tubal 36, 68 or 72junction (UTJ), distal h post-induction isthmus of the (Isth),(intramuscular ovulation ampulla (Amp)injection and infundibulum of 0.03 mg (Inf) after 10, 24, 36, 68 or gonadotropin-releasing 72 h post-induction hormone) and natural mating (n = 3/collection of the ovulation (intramuscular injection time). Fold changesof 0.03 mg relative gonadotropin-releasing to 10 h post-mating group hormone) and Different are shown. natural mating letters(n = 3/collection (a–c) time). Folddifferences represent statistical changes relative between to groups (p < 0.05). Median 10 h post-mating group are(minimum, maximum). shown. Different letters (a–c) represent statistical differences between groups (p < 0.05). Median (minimum, maximum). 3.3. Spatial Gene Expression Triggered by Natural Mating and Sperm-Free Seminal Plasma Infusion 3.3. Spatial Gene analysis The tissue Expression Triggered by expression Naturalsignificant showed Mating anddifferences Sperm-Freein Seminal Plasma Infusion IL10, TGFβ1–3 and VEGF-A expression among tissues in the different groups included in Experiment 1 (Figure The tissue analysis expression showed significant differences in IL10, TGFβ1–3 and 4a and Figure S3). VEGF-A The infundibulum expression presented among tissues thedifferent in the IL10 expression highest groups includedinin the SP-AI and Experiment control 4a 1 (Figure groups (p < 0.05), and Figure S3). whereas the cervix presented the highest IL10 The infundibulum presented the highest IL10 expression in the SP-AI and control groups (pthe expression in the NM group (p < 0.05). In case < 0.05), of TGFβ1the whereas expression, the endocervix cervix presented presented the highest the highest IL10 expression in expression the NM group in the (p NM group < 0.05). (p < In the 0.05). case of The TGFβ1distal isthmus the expression, presented the highest endocervix TGFβ2 presented expression the highest in all groups expression in the included NM group in (p Experiment < 0.05). The1 Animals 2020, 10, x 8 of 14 Animals 2020, 10, 2207 8 of 14 distal isthmus presented the highest TGFβ2 expression in all groups included in Experiment 1 (p < 0.05). Similarly, the oviductal tissues presented the highest TGFβ3 expression in the SP-AI and control (p < 0.05). Similarly, the oviductal tissues presented the highest TGFβ3 expression in the SP-AI and groups (p < 0.05). However, the proximal uterus presented the highest TGFβ3 expression in the NM control groups (p < 0.05). However, the proximal uterus presented the highest TGFβ3 expression in the group (p < 0.01). The oviductal tissues presented the highest VEGF-A expression in the SP-AI and NM group (p < 0.01). The oviductal tissues presented the highest VEGF-A expression in the SP-AI and control groups (p < 0.05), whereas the uterine tissues presented the highest VEGF-A expression in the control groups (p < 0.05), whereas the uterine tissues presented the highest VEGF-A expression in the NM group (p < 0.05). NM group (p < 0.05). U Ut Pr Ut ox p TJ vx t th Am is f In Is C D IL10 U Ut Pr t tU p ox TJ vx th Am is f In Is C D 20 h_C IL10 20 h_SP-AI 20 h_NM TGF 1 20 h_C TGF 1 20 h_SP-AI 20 h_NM 20 h_C TGF 2 TGF 2 20 h_SP-AI 20 h_NM 20 h_C TGF 3 20 h_SP-AI TGF 3 20 h_NM 20 h_C VEGF-A 20 h_SP-AI 20 h_NM VEGF-A -1 0 1 2 Row Z-Scores Figure 4. Heatmap composition of the gene expression changes among tissues in the different groups Figure 4. Heatmap composition of the gene expression changes among tissues in the different groups included in the study. Changes in IL10, TGFβ1, TGFβ2, TFGβ3 and VEGF-A expression among included in the study. Changes in IL10, TGFβ1, TGFβ2, TFGβ3 and VEGF-A expression among tissues tissues (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf) (a) at 20 h after the induction of ovulation isthmus, Isth; ampulla, Amp; and infundibulum, Inf) (a) at 20 h after the induction of ovulation with with 0.03 mg of gonadotropin-releasing hormone (GnRH) intramuscularly, as the control of ovulation 0.03 mg of gonadotropin-releasing hormone (GnRH) intramuscularly, as the control of ovulation (20 (20 h_C); 20 h post-GnRH stimulation and sperm-free seminal plasma vaginal infusion (20 h_SP-AI); h_C); 20 h post-GnRH stimulation and sperm-free seminal plasma vaginal infusion (20 h_SP-AI); and and 20 h post-GnRH stimulation and natural mating (20 h_NM); (b) at 10, 24, 36, 68 and 72 h 20 h post-GnRH stimulation and natural mating (20 h_NM); (b) at 10, 24, 36, 68 and 72 h post-GnRH- post-GnRH-stimulation and natural mating. Row Z-scores of the mean fold change relative to the stimulation and natural mating. Row Z-scores of the mean fold change relative to the reference group reference group (UTJ) are shown. Red indicates upregulation, and green indicates downregulation. (UTJ) are shown. Red indicates upregulation, and green indicates downregulation. 3.4. Spatial Gene Expression at 10 h up to 72 h Post-Mating 3.4. Spatial Gene Expression at 10 h up to 72 h Post-Mating The tissue analysis expression showed significant differences in IL10, TGFβ1–3 and VEGF-A The tissue expression amonganalysis expression tissues in showed the different groupssignificant included differences in IL10, in Experiment TGFβ1–3 2 (Figure 4b andand VEGF-A Figure S4). expression among tissues in the different groups included in Experiment 2 (Figure 4b and At 10 h post-mating (time of ovulation), the infundibulum presented the highest IL10 expression Figure S4). At 10 h post-mating (time of ovulation), the infundibulum presented the highest IL10 expression (p < Animals 2020, 10, 2207 9 of 14 (p < 0.01). Conversely, at 24 h post-mating, the endocervix presented the highest IL10 expression (p < 0.05). At 36, 68 and 72 h post-mating, the oviductal segments presented higher IL10 expression compared to the uterine tissues (p < 0.05). At 10 h post-mating, the infundibulum presented the highest TGFβ1–3 expressions (p < 0.05). The TGFβ1 expression was higher in the uterine tissues compared to the oviduct segments (p < 0.05), whereas the TGFβ2 expression was higher in the oviduct segments compared to the uterine tissues (p < 0.05). Both uterine and oviductal segments presented the highest TGFβ3 expressions (p < 0.05). The uterine tissues presented higher VEGF-A expressions (p < 0.05) compared to oviduct segments. However, at 10 and 36 h post-mating, the infundibulum presents the highest VEGF-A expression (p < 0.05). 4. Discussion The results showed a differential expression triggered by sperm-free SP infusion and natural mating on anti-inflammatory cytokines and angiogenesis-related genes, as well as the differential expression produced at different times post-mating in the rabbit, an induced ovulatory species. Additionally, the study provides the differential expression on anti-inflammatory cytokines and angiogenesis-related genes along the doe reproductive tract triggered by natural mating and sperm-free SP infusion, as well as by preimplantation embryo at different times post-mating. The SP interacts with the female reproductive tract-inducing female immune adaptation processes required to tolerate male antigens suppressing inflammation and immune rejection responses [10]. This maternal immune tolerance is essential for ongoing pregnancy success [39]. Additionally, the SP induces molecular and cellular modifications in the uterus and in the highest reproductive tract promoting embryo development and implantation competence [10]. In this regard, different soluble components present in the SP play key roles in these processes, such as the TGFβs, which can drive immune cells to tolerogenic phenotypes [40]. This study revealed differences between sperm-free SP and natural mating (that also contains SP) in the IL10, TGFβ1, TGFB3 and VEGF-A expression. The data suggest that natural mating, at 20 h post-exposure, downregulated the IL10 expression in the utero-tubal junction, probably related to the spermatozoa selection or clearance in this region. Conversely, the TGFβ1, TGFβ3 and VEGF-A expressions were upregulated in the uterine tissues in the natural mating group, whereas upregulated in the infundibulum in the sperm-free SP infusion group. Even though the SP appears to not reach the upper segments of the oviduct after mating, it seems that the signaling originated in lower segments travel to upper segments in response to SP [10]. The range of effects produced by SP in different species includes clearance of microorganisms in the uterus after mating, sperm selection, induction of ovulation, the formation of corpus luteum and supporting the development of the preimplantation embryo, among others [10]. Our observations indicate that the sperm-free SP infusion increased the expression of TGFβ1, TGFβ3 and VEGF-A in the infundibulum, whereas the expression of these genes in the natural mating group, also containing SP, remained unaltered compared to the control group. Moreover, the natural mating group increased VEGF-A expression in the distal uterus, but not in the oviduct, suggesting that the rabbit uterus reacts to post-mating inflammation [15], likely to process seminal material and recovery for uterine tissue homeostasis after mating. Our data suggest that the responsiveness of the doe reproductive tract differs between the sperm-free SP and sperm-containing SP treatments. The signaling originated by the sperm-free SP infusion was able to travel to upper segments of the oviduct, as previously demonstrated in several species [10], whereas the signal generated by the natural mating group remained in the uterine tissues. This pattern was also observed in the temporal tracking experiment of the study, suggesting that the rabbit uterus may react to the early stages of embryo development before its arrival from the oviduct, but the mechanism involved is yet not fully understood. The TGFβs isoforms present a diverse range of functions in reproductive tissues, including gonad development, gamete production, embryo implantation, fetal and placental development, development of secondary sex organs and immune tolerance of gametes and conceptus antigens [22]. The effects of Animals 2020, 10, 2207 10 of 14 TGFβs are mediated by antigen-presenting cells such as macrophages and dendritic cells, creating and maintaining an immunotolerant environment for the conceptus antigens if an anti-inflammatory dominance is established [22]. In this regard, our results revealed an upregulation of TGFβs expression at 20 h post-mating in the endometrium, suggesting that mating induces suppression of anti-sperm immune response [39]. Similar results were found at 10 h post-mating in oviductal segments, probably related to the ovulation that takes place 10 h after coitus or GnRH stimulation [7] or the suppression of anti-sperm immune response induced by the transport of spermatozoa through the oviduct before fertilization [41,42]. However, the upregulation of TGFβs expression in the oviduct was not present in the rest of the times after mating compared to 10 h post-mating, when the early embryo stages of development are present along the oviduct [43]. Thus, the effect produced by semen on the TGFβs expression changes seemed to be stronger than the effect of the embryo transport through the oviduct at different stages of development, presumably due to the different number of cells in contact with the female reproductive tract (300 million spermatozoa [44] vs. 4–12 embryos [45]). Although the effects of spermatozoa and the early embryo stages of development on TGFβs expression seems to differ in magnitude, tissue analyses revealed a TGFβ2 and TGFβ3 expression pattern in the oviduct, increasing the expression of those genes. Moreover, the TGFβ1 expression pattern in the uterus in the presence of spermatozoa and the early stages of embryo development may indicate that both antigenic compounds induced an anti-inflammatory response creating and maintaining an immunotolerance environment in the oviduct and uterus, essential for the pregnancy success [39]. Similarly, this immunotolerant environment to spermatozoa may be observed by the upregulation of the IL10 expression in the endocervix and distal uterus at 10 and 24 h post-mating. The tissue and temporal analyses revealed an interesting pattern along the oviduct during the preimplantation embryo transport. When fertilization occurs in the ampulla [46], embryos are delayed at the ampulla–isthmus junction until 48 h post-mating and then pass through the isthmus portion after 70 h post-mating to finally reach the utero-tubal junction and enter the uterus [43]. Our observations showed an upregulation of TGFβs expression in the infundibulum at the time of induced ovulation (10 h post-mating), similarly, as the pattern observed in the IL10 expression at the same time, might relate with the inflammation process associated with ovulation [47]. At 24 h post-mating, the endocervix displayed the highest expression of IL10, TGFβ1, TGFβ3 and VEGF-A, whereas the TGFβ2 expression was higher in the isthmus compared to the endocervix. Although fertilization takes place at 12–13 h post-mating [46], this upregulation of anti-inflammatory cytokines and angiogenesis mediators in the endocervix may be related to preserving spermatozoa viability and fertilization competence [13], in accordance with previous research that proposed the cervix as the first anatomical barrier for spermatozoa reservoir in rabbits [20,48,49]. The coordination between the preimplantation embryos and the differentiation of the uterus to a receptive state must be closely synchronized for successful embryo implantation [50]. Following the stromal and epithelial cell changes, the endometrial vascular bed undergoes a marked expansion before implantation [50,51]. This vascular expansion, in size and number, is markedly enhanced during implantation and placental structure development [51]. In the rabbit, the vascular supply during pregnancy is produced by the addition of new vessels and the expansion of the existing ones [52]. Our results revealed that VEGF-A expression was upregulated at 10 h post-mating in the infundibulum and ampulla, probably related to the ongoing processes like ovulation [7] and the imminent fertilization [46]. The VEGF-A expression displayed higher relative expression values at 20 h post-mating in the endometrium compared to the oviduct, perhaps related to preparing the receptive endometrium for the embryo implantation. However, this effect was not observable at different times post-mating, suggesting that additional factors, other than the analyzed in our experimental setup, are involved in the differentiation of the uterus to a receptive state before embryos reach the uterus. Animals 2020, 10, 2207 11 of 14 5. Conclusions Our results confirmed a differential modulation of the expression of anti-inflammatory and angiogenesis-related-genes by sperm-free SP and natural mating. Moreover, the gene expression analysis up to 72 h after induction of the ovulation might suggest a concerted effect of the presence of sperm and preimplantation embryos in the differential expression of these genes along the reproductive tract of the doe. Our findings warrant further research both to discriminate the effects of mating, spermatozoa, SP and preimplantation embryos on the reproductive tract of the doe and to fully describe the mechanism involved in such differential gene expression. Thus, mimicking the gene modulations induced by natural mating and sperm-free SP using physiological strategies in the current rabbit breeding systems may enhance the efficiency, productivity and sustainability of such systems. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/10/12/2207/s1, Figure S1: Changes in (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ1 and (e) VEGF-A expression among different treatments at 20 h post-treatment: 20 h post-induction of the ovulation, control (20 h_C); 20 h post-seminal plasma infusion, 20 h_SP; and 20 h post-natural mating, 20 h_NM. Tissue anatomical regions of the rabbit female reproductive tract (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf).-fold changes relative to the reference group (20 h_C) are shown. Different letters (a–c) represent statistical differences between tissues (p < 0.05). Median (minimum, maximum); Figure S2: Changes in (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ1 and (e) VEGF-A expression at different times (from 10 to 72 h) post-mating: 10, 24, 36, 68 and 72 h post-natural mating. Tissue anatomical regions of the female rabbit reproductive tract (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf).-fold changes relative to the reference group (10 h post-mating) are shown. Different letters (a–c) represent statistical differences between tissues (p < 0.05). Median [minimum, maximum]; Figure S3: Changes in (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ1 and (e) VEGF-A expression among different tissues at 20 h post-treatment. 20 h post-induction of the ovulation, control (20 h_C); 20 h post-seminal plasma infusion, 20 h_SP; and 20 h post-natural mating, 20 h_NM. Tissue anatomical regions of the female rabbit reproductive tract (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf).-fold changes relative to the reference group (UTJ) are shown. Different letters (a–d) represent statistical differences between tissues (p < 0.05). Median [minimum, maximum]; Figure S4: Changes in (a) IL10, (b) TGFβ1, (c) TGFβ2, (d) TGFβ1 and (e) VEGF-A expression among different tissues in the period 10–72 h (10, 24, 36, 68 and 72 h post-natural mating). Tissue anatomical regions of the female rabbit reproductive tract (endocervix, Cvx; distal uterus, DistUt; proximal uterus, ProxUt; utero-tubal junction, UTJ; distal isthmus, Isth; ampulla, Amp; and infundibulum, Inf).-fold changes relative to the reference group (UTJ) are shown. Different letters (a–d) represent statistical differences between tissues (p < 0.05). Median [minimum, maximum]. Author Contributions: Conceptualization, H.R.-M. and M.Á.-R.; methodology, M.L.-B.; software, J.G., A.J.-M. and M.Á.-R.; validation, A.J.-M.; formal analysis, J.G., A.J.-M. and M.Á.-R.; investigation, J.G. and C.A.M.; resources, H.R.-M., M.L.-B. and M.Á.-R.; data curation, J.G.; writing—original draft preparation, J.G.; writing—review and editing, A.J.-M., C.A.M., H.R.-M., M.L.-B. and M.Á.-R.; visualization, J.G.; supervision, M.L.M and M.Á.-R.; project administration, M.Á.-R.; funding acquisition, H.R.-M., M.L.-B. 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 (Vetenskaprådet, VR; project 2015-05919) and Juan de la Cierva Incorporación Postdoctoral Research Program (MICINN; IJDC-2015-24380). J.G. is supported by the Generalitat de Catalunya, Agency for Management of University and Research Grants co-financed with the Eropean Social Found (grants for the recruitment of new research staff 2018 FI_B 00236). Acknowledgments: We thank Annette Molbaek and Asa Schippert, from the Genomics Core Facility at LiU for expert assistance when running the bioanalyzer. We appreciate the kind support provided by Míriam Piles and Oscar Perucho and the technical staff from Torre Marimon—Institut de Recerca i Tecnologia Agroalimentàries (IRTA, Caldes de Montbui, Barcelona, Spain) and Annaïs Carbajal, Sergi Olvera-Maneu and Mateo Ruiz-Conca for their kind assistance in sample handling. 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. Animals 2020, 10, 2207 12 of 14 References 1. 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