- Research article
- Open Access
Conserved gene expression in sperm reservoirs between birds and mammals in response to mating
- Mohammad Atikuzzaman1,
- Manuel Alvarez-Rodriguez1,
- Alejandro Vicente-Carrillo1,
- Martin Johnsson2,
- Dominic Wright2 and
- Heriberto Rodriguez-Martinez1Email authorView ORCID ID profile
- Received: 30 June 2016
- Accepted: 11 January 2017
- Published: 18 January 2017
The Erratum to this article has been published in BMC Genomics 2017 18:563
Abstract
Background
Spermatozoa are stored in the oviductal functional sperm reservoir in animals with internal fertilization, including zoologically distant classes such as pigs or poultry. They are held fertile in the reservoir for times ranging from a couple of days (in pigs), to several weeks (in chickens), before they are gradually released to fertilize the newly ovulated eggs. It is currently unknown whether females from these species share conserved mechanisms to tolerate such a lengthy presence of immunologically-foreign spermatozoa. Therefore, global gene expression was assessed using cDNA microarrays on tissue collected from the avian utero-vaginal junction (UVJ), and the porcine utero-tubal junction (UTJ) to determine expression changes after mating (entire semen deposition) or in vivo cloacal/cervical infusion of sperm-free seminal fluid (SF)/seminal plasma (SP).
Results
In chickens, mating changed the expression of 303 genes and SF-infusion changed the expression of 931 genes, as compared to controls, with 68 genes being common to both treatments. In pigs, mating or SP-infusion changed the expressions of 1,722 and 1,148 genes, respectively, as compared to controls, while 592 genes were common to both treatments. The differentially expressed genes were significantly enriched for GO categories related to immune system functions (35.72-fold enrichment). The top 200 differentially expressed genes of each treatment in each animal class were analysed for gene ontology. In both pig and chicken, an excess of genes affecting local immune defence were activated, though frequently these were down-regulated. Similar genes were found in both the chicken and pig, either involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12).
Conclusion
Despite being phylogenetically distant, chicken and pig appear to share some gene functions for the preservation of viable spermatozoa in the female reservoirs.
Keywords
- Oviduct
- Sperm reservoir
- Microarray
- Bioinformatics
- Chicken
- Pig
Background
Pigs and poultry are taxonomically distant animal species differing in reproductive anatomy and physiology. However, they both share internal fertilization, e.g. the capacity to store immunologically foreign spermatozoa in the female genital tract during the interval between mating and ovulation, this either being short (30–36 h in the case of the pig, with multiple ovulations over a 30 min period) or long (over several weeks, including recurrent daily ovulations as in modern, in the case of highly selected egg-layer poultry) [1–3]. The utero-tubal junction (UTJ) of the pig and the utero-vaginal junction (UVJ) of the chicken oviduct are analogous, a location where a subpopulation of spermatozoa are selectively stored post-mating, remaining alive and potentially fertile [1–3], before being gradually released for the fertilization of ovulated eggs [4–6]. In mice, the presence of spermatozoa in the oviduct leads to changes in gene expression, with upregulation of adrenomedullin and prostaglandin-endoperoxide synthase 2 transcripts [7]. Likewise, mating changes gene expression in the UVJ of the ancestral Red Junglefowl [8] as well as in an advanced intercross line (AIL, crossing between Red Junglefowl and White Leghorn chicken, [9]). Insemination has been reported as being capable of increasing mRNA expression of transforming growth factor beta (TGFβs) and TGFβ receptors (TβRs) but of decreasing mRNA expression of interleukin 1 beta (IL1B) and lipopolysaccharide induced TNF factor (LITAF) in the UVJ, which has also being implicated in the survival of sperm-storage tubuli (SST)-resident spermatozoa [10–12]. Studies in pigs have to date solely focused on the area of the oviduct where fertilization takes place [13–15]. Thus, in contrast to avian studies, trials in mammals have yet to examine the sperm reservoir areas.
Birds and pigs differ in internal genital tract anatomy, with chickens lacking accessory sexual glands. In contrast, the boar has a complete set of accessory glands whose concerted secretions form the seminal plasma; an heterogeneous fluid that accompanies the spermatozoa -embedded in the intraluminal cauda epididymis fluid- when emitted at ejaculation. In either animal class, semen is an immunologically foreign cell-suspension for the female, which should promptly elicit an immune response to eliminate it. It has been hypothesized that semen signals a genomic shift in the oviduct of the female that modulates the expression of genes involved in immune processes in both chickens [9–12, 16] and mammals [13–15], resulting in a state of immune tolerance during the lengthy storage of spermatozoa [17]. However, whether these divergent animal classes share a common mechanism is unclear.
Moreover, whether it is the entire semen (e.g. both the spermatozoa and the seminal fluid), the spermatozoa themselves or the cell-free seminal fluid that elicit such changes in gene expression in the sperm reservoirs is, to the best of our knowledge, poorly explored, with the exception of studies performed in Red Junglefowl [8] and mutant mice whose ejaculates were sperm-free [7]. The protein composition of seminal fluid has been extensively studied in chickens [18–20] and mammals [21–24] including the pig [25, 26]. In mammals, seminal plasma proteins are considered the most relevant for fertility [27–30], presumably owing to the induction of an initial but transient inflammation to clear microorganisms, superfluous gametes and proteins from the genital tract [26], followed by the induction of an immunological tolerance to paternal alloantigens via the expansion of regulatory T cells [22] following endometrial synthesis of toll-like receptor 4 (TLR4)-regulated cytokines and chemokines [24].
In chickens, the seminal fluid contains a few proteins classified as immune regulatory and/or defense such as gallinacin-9, ovotransferrin, serum albumin, thioredoxin, and peroxiredoxin-6 [18, 19]. Our own studies also indicate that the levels of immune-modulatory cytokines TGFβ2 and CXCL10 in the seminal fluid as well as the expression of the proteins Gallinacin-9 and Ig lambda chain C differed between low- and high egg-laying chickens [31], which might be related to sperm survival capacity in the female oviduct. This relationship depends on the function of the sperm reservoirs, which are highly correlated with fertility in both chickens [12] and pigs [2]. Modern domestic chickens and pigs are considered highly fertile. The modern layer poultry White Leghorn lays around 300 eggs per year, and a modern Swedish Landrace female pig produces over 26 live piglets per year. However, whether the oviduct sperm reservoirs respond to the entry of semen or SF/SP by a change in gene expression, and whether the response is similar between such different animal species, is yet to be tested.
In this study it is hypothesized that zoologically distant modern pigs and poultry, despite being selected for productivity (litter size or egg-laying rate, among other variables), share conserved mechanisms to tolerate the lengthy presence of immunologically-foreign spermatozoa in the oviduct sperm reservoirs. To test this hypothesis, microarray analyses on the functional sperm reservoir tissues of White Leghorn hens and Swedish Landrace sows were performed to identify gene expression changes in UVJ and UTJ after mating (entire semen deposition) or in vivo artificial infusion with sperm-free seminal fluid.
Methods
Experimental design
Gene expression analyses of the functional oviduct sperm reservoir (UVJ in chickens and UTJ in pigs) were performed in twelve modern White Leghorn breed female chickens (Gallus gallus domestica, Experiment 1) and twelve modern Swedish Landrace female pigs (Sus scrofa domestica, Experiment 2). The females of either species were allotted to one of three separate groups: a natural mating group (n = 4), where females (hen or sow) were mated to a single male each; sperm-free SF/SP inseminated group (n = 4) where females were artificially inseminated with pooled seminal fluid/plasma collected from the same males used for the mating group, and finally a control group (n = 4) of females that were neither mated nor inseminated. The oviduct reservoirs were collected post-mortem (UVJ, hens) or surgically (UTJ, sows) 24 h after treatments along with control animals. The tissues were either investigated for gene expression using custom-made chicken microarray (Roche NimbleGen, 12X 135 k array) or porcine gene chip microarray (Affymetrix, Inc. 3420 Central Expressway, Santa Clara, CA 95051, USA).
Animal husbandry and experimental handling were performed in compliance with the European Community (Directive 2010/63/EU) and current Swedish legislation (SJVFS 2015:24). Throughout all experiments, animals were handled carefully and in such a way as to avoid any unnecessary stress. The experiments were approved in advance by the “Regional Committee for Ethical Approval of Animal Experiments” (Linköpings Djurförsöksetiska nämnd) in Linköping, Sweden (permit no 75–12).
Semen evaluation
Sperm concentration and motility were evaluated using a light microscope (Zeiss, Stockholm Sweden) equipped with a thermal plate (41 °C for chicken semen or 38 °C for pig semen), positive phase contrast optics (10x objective), a Charge Coupled Device (CCD) camera (UI-1540LE-M-HQ, Ueye, IDS Imaging Development Systems GmbH, Ubersulm, Germany), and the Qualisperm® Software (Biophos SA, Lausanne, Switzerland).
Experiment 1
Experimental birds
A White Leghorn (WL) layer breed selected for high food conversion efficiency and commonly commercial bred for egg-production [32] was used. The details of the chicken rearing are described in Johnsson et al. 2012 [33]. Briefly, all chickens were kept separated by gender at the facilities of Linköping University (LiU). Food, water and perches were available ad libitum and chicken were held under controlled temperature and light regimes (12 h:12 h light/dark cycle) in 1–2 m2 pens depending on age for their first seven weeks.
Collection of semen, evaluation, mating and artificial insemination of seminal fluid
Chickens were subjected to semen collection and evaluation following the same procedure as our previous study [9]. Briefly, semen was collected by manual abdominal massage and was primarily extended with Dulbecco’s medium and examined in four replicates for sperm concentration and kinematics using a light microscope as described above. Only males yielding semen of high quality (sperm numbers and proportions of progressively motile spermatozoa, evaluated using the instrumentation detailed above) were selected for mating/insemination. Four hens were individually paired with males of proven fertility -using one male per hen (treatment 1). The collected semen from selected males was also subjected to centrifugation at 21,000 x g at 4 °C for 10 min. The supernatant (SF) was harvested and ejaculates pools (1 pool/male to make 4 individual pools) were made from four males used for the mating program. A 200 μl aliquot of pooled SF was inseminated into the cloaca using a plastic Pasteur pipette (Treatment 2). Four hens were left unmated or un-inseminated as controls.
Collection of UVJ
All hens (treatment 1 and 2) were euthanized by cervical dislocation followed by decapitation, 24 h after mating or insemination, along with the control hens. Immediately post-mortem, the oviduct segments were identified and dissected out under stereomicroscopy. The UVJ containing the SST was then collected using disposable razor blades, following classical descriptions [34] and snap-frozen in liquid nitrogen (LN2), prior to storage at −80 °C until further processed. A supplementary UVJ containing SST sample per mated hen was also fixed in 4% paraformaldehyde for histological confirmation of sperm presence in the SST-reservoirs. The confirmation of the presence of sperm was performed prior to the use of the UVJ tissues from mated or SF-infused or control hens in the microarray experiment.
Microarrays hybridization and scanning
Total RNA extraction (using Trizol), integrity evaluation, cDNA synthesis and custom-made microarray analysis (Roche NimbleGen Systems, Inc., Madison, WI, USA) were done following Atikuzzaman et al. 2015 [9]. A total of 12 microarrays (4 arrays per group) were run in this experiment.
Experiment 2
Experimental pigs
Young mature boars (n = 5) of proven sperm quality (concentration, morphology and motility) and weaned sows (parity 1–3, n = 12) of the Swedish Landrace breed were recruited from a controlled breeding farm and individually kept in separate pens at the Translational Medicine Center (TMC/CBR-3) of Linköping University under controlled temperature and light regimes (12 h:12 h light/dark cycle). Pigs were fed with commercial feedstuff (Lantmännen, Stockholm, Sweden) according to national standards [35], provided with water ad libitum and with all animals receiving the same management.
Semen collection, evaluation and harvesting of seminal plasma
Semen was manually collected (gloved-hand method) weekly. Only ejaculates with at least 70% motile and 75% morphologically normal spermatozoa immediately after collection were used. Seminal plasma (SP) was harvested from the whole ejaculate after double centrifugation at 1,500xg for 10 min. The harvested crude-SP was kept at −20 °C, until use.
Detection of oestrus
The females were observed two times daily for pro-oestrus and oestrus behavioural signs while holding snout contact with a neighbouring boar, by the application of backpressure by experienced personnel. Animals that showed a standing oestrous reflex were considered to be in oestrus and were used in the experiments. Sows were randomly allotted to a control group (n = 4, unmated/non-inseminated), mated (Treatment 1, n = 4) or SP-inseminated (Treatment 2, n = 4).
Mating and insemination with seminal plasma
Sows were, on the first day of behavioral oestrus, either cervically inseminated (disposable AI-catheter, Minitüb, Munich, Germany) with 50 ml of Beltsville Thawing Solution (BTS, Control group); mated with a boar (Treatment 1 group) or artificially inseminated with 50 ml of SP (Treatment 2 group).
Collection of tissues
On the second day of standing oestrus (pre/periovulation) the sows were sedated by the administration of a mixture of 5 mg dexmedetomidine (Dexdomitor, Orion Pharma Animal Health, Sollentuna, Sweden) and 100 mg tiletamine hydrochloride/zolazepam hydrochloride (Zoletil vet, Virbac A/S, Kolding, Denmark) intramuscularly. General anesthesia was induced using sodium thiopental (Abbott Scandinavia AB, Solna, Sweden) 7 mg/kg body weight, intravenously, and was maintained with isoflurane (Baxter Medical AB, Kista, Sweden, 3.5-5%) administered via a tracheal cuffed tube by a close-circuit PVC-ventilator (Servo ventilator 900 D, SIEMENS-ELEMA AB, Solna, Sweden). Peripheral blood was collected (Vacutainer containing K2EDTA, Greiner Bio-One GmbH, Kremsmünster, Austria) centrifuged at 300 x g for 10 min at room temperature. The blood plasma was harvested and stored at −20 °C until analysed for oestradiol (E2) and progesterone (P4) concentrations. The left and right UTJ were exposed by mid-ventral incision. The complete UTJ of each side was removed immediately after clamping the irrigating blood vessels, being longitudinally divided into two equal pieces. One of the pieces was plunged in liquid nitrogen (LN2) and later stored at −80 °C while the other piece was fixed in 4% paraformaldehyde for histological confirmation of sperm presence. The confirmation of presence or absence of spermatozoa was done prior to use the UTJ tissues from mated or SP-infused or control sows for microarray experiment. The ovaries were photographed and the follicles visually counted. There was a mean of 22.30 ± 7.29 (mean ± standard deviation) follicles per sow, without significant differences between sow-groups.
Determinations of oestradiol and progesterone concentrations
Concentrations of oestradiol (E2) and progesterone (P4) were measured in individual blood plasma (50 μl) using porcine enzyme linked immune sorbent assay (ELISA) kits (Cat#MBS700342 and Cat#MBS703577, MyBiosource Inc., San Diego, CA, USA), after preparation of a standard curve for the individual hormones, following the manufacturer protocol. The optical density of each microplate well was determined using a microplate reader (TECAN, Sunrise GmbH, Grödig, Austria) set at 450 nm. Oestradiol concentrations (mean ± SD in pg/ml) were 376.50 ± 27.76 in controls, 349.10 ± 62.19 in mated and 294.20 ± 80.24 in SF-inseminated sows and those of progesterone (mean ± SD in ng/ml) were <0.68 ± 0.34 without significant differences between sow groups, confirming the animals were in pre/peri-ovulatory oestrus.
Microarrays hybridization and scanning
Total RNA was extracted using Trizol from UTJ samples and evaluated following the protocol used in Atikuzzaman et al. 2015 [9]. Equal amounts of total RNA (250 ng) from each UTJ were used to make cDNA using GeneChip® WT PLUS reagent kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer protocol. Finally, 3.5 μg of fragmented and labelled single stranded complementary DNA (41 μl) was mixed with 109 μl of hybridization master mix to make a cocktail hybridization mix for a single reaction. The hybridization cocktail was then incubated first at 99 °C for 5 min, followed by a descent to 45 °C until loading on the array chip (Porcine gene 1.0 ST GeneChip® Cartidge Array, Affymetrix). A total of 130 μl of the cocktail hybridization mix was loaded into the array chip and they were incubated at 45 °C under rotation at 60 revolutions per minute for 16 h. The hybridized cartridge array chip was then unloaded and subjected to washing and staining using a GeneChip® Fluidics Station 450 (Affymetrix), to be finally scanned using the Affymetrix GeneChip® scanner GCS3000.
Microarray data analysis and bioinformatics
The expression data of experiments 1 and 2 were processed using the Robust Multichip Average (RMA) normalization procedure, computing average expression values by background adjustment, quantile normalization between arrays, and summarization, as implemented in the oligo package of Bioconductor/DEVA Software (Roche NimbleGen, Inc, DEVA 1.2.1). The statistical analysis of the normalized gene expression data was performed using the open source RStudio package (RStudio, Inc. Version 0.98.507). Linear models using the empirical Bayes’ approach as implemented in the package ‘limma’ were used to calculate differentially expressed transcripts. Two different multiple testing corrections were applied. The first was a Benjamini-Hochberg False Discover Rate (FDR) correction [36], whilst the second was based on a permutation test. The permutation test was used in addition to the FDR test, given that a number of the custom probes used on the microarray (specifically those based on EST transcripts) were replicates of genes already represented on the array, thus the FDR threshold maybe overly restrictive. The permutation test was performed using the Limma package by randomising the class classifications, then calculating gene expression differentiation globally, before retaining the top 1% value. This was repeated 1,000 times, before the top 5% of permuted values were then used as an experiment-wide threshold (with this p-value corresponding to a nominal value of approximately p < 0.002). The redundant and uncharacterized transcripts were excluded from the list after both the multiple testing corrections to make a final list of differentially expressed genes. An enrichment analysis of these differentially expressed genes (both permuted and non-permuted) was performed via a statistical overrepresentation test for gene ontology (GO) biological process, comparing the total number of reference genes in the genome of Gallus gallus (15,789) and of Sus scrofa (21,398) using the Panther Classification System for GO [37]. The top 200 of these differentially expressed genes (100 upregulated and 100 downregulated genes based on the log fold change at P < 0.05) in both animal classes were selected for further bioinformatic analyses. The GOs of the top differentially expressed genes were analysed under the PANTHER GO-Slim Biological Process category. PANTHER extracted differentially expressed genes in both animal classes and those in the GO-term category of immune system process were then searched for functional pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [38]. Additional molecular functions of these differentially expressed immune system process genes were extracted from the protein knowledge base of the UniProt Consortium [39].
Results
Both mating and insemination of sperm-free SF/SP elicited changes in gene expression in the sperm reservoirs of chicken and pig oviducts
Volcano plots depicting differentially expressed probes for oviductal sperm reservoirs in chicken (UVJ) and pig (UTJ), following mating or sperm-free SF/SP-infusion. The x-axis represents the fold change and the y-axis represents the statistical significance (−log10 of P. value). Each of the oligonucleotide probes is represented by a single dot. The red dots represent log fold change > 1 or < −1 at p-value <0.05. The green dots represent log fold change >1 or < −1 at FDR adjusted p-value <0.05. The dots above the horizontal broken lines are probes that were differentially expressed at p-vaue <0.05. a) Comparison between mating (n = 4) and control (n = 4) group chicken, b) comparison between SF (n = 4) and control (n = 4) group chicken, c) comparison between mating (n = 4) and control (n = 4) group pigs and d) comparison between SF (n = 4) and control (n = 4) group pigs
Over- and under-represented mating-induced differentially expressed genes (P < 0.05) in the GO- biological process involving immune system function in chicken
GO-BP (immune function) | REF | DE (221) | EXPC | FE(+/−) | P value |
|---|---|---|---|---|---|
CD8-positive, gamma-delta intraepithelial T cell differentiation (GO:0002305) | 2 | 1 | 0.03 | +35.72 | 2.76E-02 |
Gamma-delta intraepithelial T cell differentiation (GO:0002304) | 2 | 1 | 0.03 | +35.72 | 2.76E-02 |
Negative regulation of monocyte chemotaxis (GO:0090027) | 2 | 1 | 0.03 | +35.72 | 2.76E-02 |
Immunoglobulin secretion (GO:0048305) | 2 | 1 | 0.03 | +35.72 | 2.76E-02 |
T-helper 1 cell activation (GO:0035711) | 2 | 1 | 0.03 | +35.72 | 2.76E-02 |
Gamma-delta T cell activation (GO:0046629) | 4 | 2 | 0.06 | +35.72 | 1.50E-03 |
Chronic inflammatory response (GO:0002544) | 3 | 1 | 0.04 | +23.81 | 4.11E-02 |
Complement activation, lectin pathway (GO:0001867) | 3 | 1 | 0.04 | +23.81 | 4.11E-02 |
Negative regulation of immature T cell proliferation (GO:0033087) | 3 | 1 | 0.04 | +23.81 | 4.11E-02 |
Gamma-delta T cell differentiation (GO:0042492) | 3 | 1 | 0.04 | +23.81 | 4.11E-02 |
Negative regulation of T cell proliferation (GO:0042130) | 24 | 2 | 0.34 | +5.95 | 4.51E-02 |
B cell proliferation (GO:0042100) | 24 | 2 | 0.34 | +5.95 | 4.51E-02 |
Regulation of T cell proliferation (GO:0042129) | 80 | 5 | 1.12 | +4.47 | 5.69E-03 |
Positive regulation of T cell proliferation (GO:0042102) | 52 | 3 | 0.73 | +4.12 | 3.73E-02 |
Regulation of T cell activation (GO:0050863) | 144 | 7 | 2.02 | +3.47 | 4.50E-03 |
Positive regulation of T cell activation (GO:0050870) | 91 | 4 | 1.27 | +3.14 | 4.00E-02 |
Positive regulation of leukocyte cell-cell adhesion (GO:1903039) | 95 | 4 | 1.33 | +3.01 | 4.56E-02 |
Regulation of lymphocyte proliferation (GO:0050670) | 120 | 5 | 1.68 | +2.98 | 2.78E-02 |
Regulation of leukocyte proliferation (GO:0070663) | 125 | 5 | 1.75 | +2.86 | 3.23E-02 |
Regulation of leukocyte activation (GO:0002694) | 249 | 8 | 3.49 | +2.3 | 2.51E-02 |
Regulation of lymphocyte activation (GO:0051249) | 219 | 7 | 3.07 | +2.28 | 3.58E-02 |
Defence response (GO:0006952) | 498 | 12 | 6.97 | +1.72 | 4.92E-02 |
Over- and under-represented sperm-free SF-induced differentially expressed genes (P < 0.05) in the GO- biological process involving immune system function in chicken
GO-BP (immune function) | REF | DE (721) | EXPC | FE(+/−) | P value |
|---|---|---|---|---|---|
Positive regulation of CD8-positive, alpha-beta cytotoxic T cell extravasation (GO:2000454) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Regulation of CD8-positive, alpha-beta cytotoxic T cell extravasation (GO:2000452) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of CD8-positive, alpha-beta T cell extravasation (GO:2000451) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Regulation of CD8-positive, alpha-beta T cell extravasation (GO:2000449) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of interleukin-15 production (GO:0032738) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of T cell extravasation (GO:2000409) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Regulation of T cell extravasation (GO:2000407) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Regulation of interleukin-15 production (GO:0032658) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of neutrophil apoptotic process (GO:0033031) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of isotype switching to IgA isotypes (GO:0048298) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
T-helper 2 cell cytokine production (GO:0035745) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
TIRAP-dependent toll-like receptor 4 signaling pathway (GO:0035665) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
TIRAP-dependent toll-like receptor signaling pathway (GO:0035664) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Positive regulation of establishment of T cell polarity (GO:1903905) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Interleukin-8 biosynthetic process (GO:0042228) | 1 | 1 | 0.05 | +21.9 | 4.46E-02 |
Negative regulation of macrophage chemotaxis (GO:0010760) | 4 | 2 | 0.18 | +10.95 | 1.48E-02 |
Positive regulation of macrophage chemotaxis (GO:0010759) | 5 | 2 | 0.23 | +8.76 | 2.24E-02 |
Regulation of macrophage chemotaxis (GO:0010758) | 11 | 4 | 0.5 | +7.96 | 1.77E-03 |
Negative regulation of leukocyte chemotaxis (GO:0002689) | 9 | 3 | 0.41 | +7.3 | 8.50E-03 |
Positive regulation of macrophage differentiation (GO:0045651) | 10 | 3 | 0.46 | +6.57 | 1.13E-02 |
Regulation of macrophage differentiation (GO:0045649) | 14 | 4 | 0.64 | +6.26 | 4.17E-03 |
Negative regulation of alpha-beta T cell activation (GO:0046636) | 12 | 3 | 0.55 | +5.47 | 1.82E-02 |
Lymphocyte chemotaxis (GO:0048247) | 14 | 3 | 0.64 | +4.69 | 2.71E-02 |
Monocyte chemotaxis (GO:0002548) | 16 | 3 | 0.73 | +4.11 | 3.79E-02 |
Negative regulation of response to cytokine stimulus (GO:0060761) | 27 | 4 | 1.23 | +3.24 | 3.66E-02 |
Positive regulation of T cell proliferation (GO:0042102) | 52 | 6 | 2.37 | +2.53 | 3.39E-02 |
Regulation of leukocyte chemotaxis (GO:0002688) | 57 | 6 | 2.6 | +2.31 | 4.89E-02 |
Regulation of leukocyte migration (GO:0002685) | 85 | 8 | 3.88 | +2.06 | 4.39E-02 |
over- and under-represented mating-induced differentially expressed genes (P < 0.05) in the GO- biological process involving immune system function in pig
GO-BP (immune function) | REF | DE (1179) | EXPC | FE (+/−) | P value |
|---|---|---|---|---|---|
Regulation of T cell mediated immune response to tumor cell (GO:0002840) | 3 | 2 | 0.17 | +12.1 | 1.22E-02 |
Regulation of macrophage apoptotic process (GO:2000109) | 5 | 2 | 0.28 | +7.26 | 3.16E-02 |
Interleukin-8 secretion (GO:0072606) | 5 | 2 | 0.28 | +7.26 | 3.16E-02 |
T-helper 17 cell differentiation (GO:0072539) | 5 | 2 | 0.28 | +7.26 | 3.16E-02 |
T-helper 17 type immune response (GO:0072538) | 5 | 2 | 0.28 | +7.26 | 3.16E-02 |
Positive regulation of mast cell chemotaxis (GO:0060754) | 6 | 2 | 0.33 | +6.05 | 4.39E-02 |
Toll-like receptor 4 signaling pathway (GO:0034142) | 13 | 3 | 0.72 | +4.19 | 3.61E-02 |
Positive regulation of monocyte chemotaxis (GO:0090026) | 14 | 3 | 0.77 | +3.89 | 4.33E-02 |
Positive regulation of leukocyte chemotaxis (GO:0002690) | 59 | 7 | 3.25 | +2.15 | 4.75E-02 |
Regulation of lymphocyte activation (GO:0051249) | 239 | 20 | 13.17 | +1.52 | 4.65E-02 |
Over- and under-represented sperm-free SP-induced differentially expressed genes (P < 0.05) in the GO- biological process involving immune system function in pig
GO-BP (immune function) | REF | DE (775) | EXPC | FE (+/−) | P value |
|---|---|---|---|---|---|
Positive regulation of antigen processing and presentation of peptide antigen via MHC class II (GO:0002588) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Positive regulation of antigen processing and presentation of peptide or polysaccharide antigen via MHC class II (GO:0002582) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Cytokine secretion involved in immune response (GO:0002374) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
B cell cytokine production (GO:0002368) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Positive regulation of IP-10 production (GO:0071660) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Regulation of IP-10 production (GO:0071658) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Positive regulation of B cell chemotaxis (GO:2000538) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Regulation of B cell chemotaxis (GO:2000537) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Positive regulation of mast cell activation by Fc-epsilon receptor signaling pathway (GO:0038097) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Tumor necrosis factor secretion (GO:1990774) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Isotype switching to IgG isotypes (GO:0048291) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
T-helper 1 cell activation (GO:0035711) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Response to TNF agonist (GO:0061481) | 1 | 1 | 0.04 | +27.61 | 3.56E-02 |
Positive regulation of interleukin-1 alpha secretion (GO:0050717) | 3 | 2 | 0.11 | +18.41 | 5.49E-03 |
regulation of T cell mediated immune response to tumor cell (GO:0002840) | 3 | 2 | 0.11 | +18.41 | 5.49E-03 |
Positive regulation of interleukin-1 alpha production (GO:0032730) | 4 | 2 | 0.14 | +13.81 | 9.52E-03 |
Regulation of interleukin-1 alpha secretion (GO:0050705) | 4 | 2 | 0.14 | +13.81 | 9.52E-03 |
Toll-like receptor 2 signaling pathway (GO:0034134) | 5 | 2 | 0.18 | +11.04 | 1.45E-02 |
Regulation of interleukin-1 alpha production (GO:0032650) | 5 | 2 | 0.18 | +11.04 | 1.45E-02 |
Interleukin-10 production (GO:0032613) | 5 | 2 | 0.18 | +11.04 | 1.45E-02 |
Positive regulation of interleukin-1 secretion (GO:0050716) | 11 | 4 | 0.4 | +10.04 | 7.60E-04 |
Regulation of type I interferon-mediated signaling pathway (GO:0060338) | 13 | 4 | 0.47 | +8.5 | 1.40E-03 |
Positive regulation of interleukin-1 beta secretion (GO:0050718) | 10 | 3 | 0.36 | +8.28 | 6.03E-03 |
Positive regulation of type I interferon-mediated signaling pathway (GO:0060340) | 7 | 2 | 0.25 | +7.89 | 2.72E-02 |
Positive regulation of interleukin-1 production (GO:0032732) | 14 | 4 | 0.51 | +7.89 | 1.83E-03 |
Interleukin-8 production (GO:0032637) | 7 | 2 | 0.25 | +7.89 | 2.72E-02 |
Regulation of interleukin-1 beta secretion (GO:0050706) | 14 | 4 | 0.51 | +7.89 | 1.83E-03 |
Regulation of interleukin-1 secretion (GO:0050704) | 18 | 5 | 0.65 | +7.67 | 5.67E-04 |
Positive regulation of interleukin-10 production (GO:0032733) | 19 | 5 | 0.69 | +7.27 | 7.21E-04 |
Positive regulation of interleukin-8 secretion (GO:2000484) | 8 | 2 | 0.29 | +6.9 | 3.47E-02 |
Positive regulation of interleukin-1 beta production (GO:0032731) | 12 | 3 | 0.43 | +6.9 | 9.89E-03 |
Positive regulation of interferon-alpha production (GO:0032727) | 12 | 3 | 0.43 | +6.9 | 9.89E-03 |
Toll-like receptor 4 signaling pathway (GO:0034142) | 13 | 3 | 0.47 | +6.37 | 1.22E-02 |
Positive regulation of interleukin-6 secretion (GO:2000778) | 13 | 3 | 0.47 | +6.37 | 1.22E-02 |
Regulation of response to interferon-gamma (GO:0060330) | 9 | 2 | 0.33 | +6.14 | 4.28E-02 |
Inflammatory response to antigenic stimulus (GO:0002437) | 14 | 3 | 0.51 | +5.92 | 1.49E-02 |
Regulation of interferon-alpha production (GO:0032647) | 14 | 3 | 0.51 | +5.92 | 1.49E-02 |
Regulation of interleukin-10 production (GO:0032653) | 26 | 5 | 0.94 | +5.31 | 2.82E-03 |
Regulation of interleukin-1 beta production (GO:0032651) | 27 | 5 | 0.98 | +5.11 | 3.30E-03 |
Regulation of interleukin-1 production (GO:0032652) | 34 | 6 | 1.23 | +4.87 | 1.69E-03 |
MyD88-dependent toll-like receptor signaling pathway (GO:0002755) | 17 | 3 | 0.62 | +4.87 | 2.46E-02 |
Cellular response to interleukin-4 (GO:0071353) | 18 | 3 | 0.65 | +4.6 | 2.85E-02 |
Positive regulation of interferon-beta production (GO:0032728) | 20 | 3 | 0.72 | +4.14 | 3.71E-02 |
Positive regulation of type I interferon production (GO:0032481) | 27 | 4 | 0.98 | +4.09 | 1.76E-02 |
Somatic diversification of immune receptors via germline recombination within a single locus (GO:0002562) | 28 | 4 | 1.01 | +3.94 | 1.98E-02 |
Positive regulation of interleukin-6 production (GO:0032755) | 42 | 6 | 1.52 | +3.94 | 4.71E-03 |
Positive regulation of interleukin-8 production (GO:0032757) | 30 | 4 | 1.09 | +3.68 | 2.47E-02 |
Regulation of interferon-beta production (GO:0032648) | 30 | 4 | 1.09 | +3.68 | 2.47E-02 |
Regulation of type I interferon production (GO:0032479) | 38 | 5 | 1.38 | +3.63 | 1.33E-02 |
Toll-like receptor signaling pathway (GO:0002224) | 40 | 5 | 1.45 | +3.45 | 1.62E-02 |
Positive regulation of tumor necrosis factor production (GO:0032760) | 34 | 4 | 1.23 | +3.25 | 3.64E-02 |
Positive regulation of tumor necrosis factor superfamily cytokine production (GO:1903557) | 35 | 4 | 1.27 | +3.16 | 3.98E-02 |
Positive regulation of cytokine secretion (GO:0050715) | 62 | 7 | 2.25 | +3.12 | 8.19E-03 |
Positive regulation of innate immune response (GO:0045089) | 90 | 10 | 3.26 | +3.07 | 1.96E-03 |
Positive regulation of adaptive immune response (GO:0002821) | 57 | 6 | 2.06 | +2.91 | 1.88E-02 |
Regulation of cytokine-mediated signaling pathway (GO:0001959) | 60 | 6 | 2.17 | +2.76 | 2.35E-02 |
Regulation of interleukin-6 production (GO:0032675) | 70 | 7 | 2.54 | +2.76 | 1.50E-02 |
Regulation of response to cytokine stimulus (GO:0060759) | 62 | 6 | 2.25 | +2.67 | 2.69E-02 |
Positive regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains (GO:0002824) | 54 | 5 | 1.96 | +2.56 | 4.85E-02 |
Regulation of innate immune response (GO:0045088) | 154 | 14 | 5.58 | +2.51 | 1.83E-03 |
Regulation of lymphocyte mediated immunity (GO:0002706) | 82 | 7 | 2.97 | +2.36 | 3.17E-02 |
Positive regulation of defense response (GO:0031349) | 155 | 11 | 5.61 | +1.96 | 2.82E-02 |
Regulation of immune effector process (GO:0002697) | 260 | 18 | 9.42 | +1.91 | 7.84E-03 |
Positive regulation of immune response (GO:0050778) | 256 | 17 | 9.27 | +1.83 | 1.39E-02 |
Regulation of inflammatory response (GO:0050727) | 185 | 12 | 6.7 | +1.79 | 4.03E-02 |
Regulation of leukocyte cell-cell adhesion (GO:1903037) | 189 | 12 | 6.85 | +1.75 | 4.59E-02 |
Regulation of defense response (GO:0031347) | 394 | 25 | 14.27 | +1.75 | 5.84E-03 |
Immune effector process (GO:0002252) | 253 | 16 | 9.16 | +1.75 | 2.46E-02 |
Regulation of immune response (GO:0050776) | 398 | 25 | 14.41 | +1.73 | 6.59E-03 |
Regulation of cytokine production (GO:0001817) | 352 | 22 | 12.75 | +1.73 | 1.09E-02 |
Innate immune response (GO:0045087) | 280 | 17 | 10.14 | +1.68 | 2.93E-02 |
Positive regulation of immune system process (GO:0002684) | 481 | 29 | 17.42 | +1.66 | 6.26E-03 |
Immune system development (GO:0002520) | 470 | 28 | 17.02 | +1.64 | 8.28E-03 |
Regulation of immune system process (GO:0002682) | 785 | 45 | 28.43 | +1.58 | 2.05E-03 |
Cytokine-mediated signaling pathway (GO:0019221) | 295 | 4 | 10.68 | - 0.37 | 1.81E-02 |
Mating or SF-infusion changed the expression of treatment-specific genes as well as of a common subset of genes in the sperm reservoir
Venn diagrams of differentially expressed genes (p < 0.05) in the oviductal sperm reservoirs of chicken (UVJ) and pig (UTJ) after mating or SF/SP-infusion, compared to their controls: a) upregulated genes in the UVJ of chicken, b) downregulated genes in the UVJ of chicken, c) upregulated genes in UTJ of pigs, D) downregulated genes in UTJ of pigs
A subset of stimulus-responsive and immune system-process genes were differentially expressed in the sperm reservoirs after mating and SF-infusion; the expression pattern differed chicken and pigs after mating but not after in vivo SF-infusion
Biological process categories of top 200 differentially expressed genes (100 upregulated and 100 downregulated) selected from each comparison (mating versus control and SF/SP-infusion versus control in each animal class). The y-axis represents number of differentially expressed genes and the x-axis represents biological process categories analyzed by PANTHER gene ontology classification database. The black and white pattern columns represent differentially expressed genes in chicken while colored columns represent differentially expressed genes in pigs. a) Comparison between mating and control group of chicken and pig, b) comparison between SF/SP-infusion and control group of chicken and pig
Differentially expressed immune-modulatory genes in the oviductal sperm reservoirs. For details see Additional file 5: Table S5
Treatment | Animals | Local Immune defense (LID) at oviduct | Differentially expressed genes in the oviductal primary sperm reservoirs |
|---|---|---|---|
Mating | Chicken | Enhance LID at UVJ | CCR9, TNFSF4, TFPI |
Suppress LID at UVJ | LHX3, MASP1, NPY6R, NRXN1, F2, PTK2, HSPA13, NELL1 | ||
Pig | Enhance LID at UTJ | GZMK, LY96, CD36, LOC100513220, PDZD2, DPP4 | |
Suppress LID at UTJ | CSMD3, DRD2, SELL | ||
SF/SP-infusion | Chicken | Enhance LID at UVJ | DLK2, CCL1, CCR4, LIF, NOX3, ASTL |
Suppress LID at UVJ | ADCYAP1R1, DLL4, BPIL3 | ||
Pig | Enhance LID at UTJ | GPR116, F8, GZMK, PTK2B, LY96, SEMA6A | |
Suppress LID at UTJ | TXNRD1, NOR-1 |
Mating or SF-infusion changed the expression of the same genes in the oviductal sperm reservoir of both chickens and pigs in some instances
Differentially expressed genes (top 200) with similar function(s) between animal classes
Treatment | Changes | DE genes in SR | Category of the DE genes | UniProt Biological process and molecular function | KEGG category | KEGG subcategory | KEGG pathways | |
|---|---|---|---|---|---|---|---|---|
Hens | Sows | |||||||
Mating | UP | SLC16A2, SLC4A9 | SLC13A1, SLC35F1 | solute carrier family | Transmembrane transporter activity | Organismal system | Endocrine system | Thyroid hormone signaling pathway |
DOWN | MMP27 | ADAMTS3, MMP3, MMP12 | metallopeptidase | Metallopeptidase activity, regulation of cell migration | Environmental information processing | Signal transduction | TNF signaling pathway | |
TBX4 | TBX20 | T-box family | Transcription factor activity | - | - | - | ||
SF/SP-infusion | UP | CDH17, CDH19 | CDH13 | cadherin family | Cell adhesion | - | - | - |
IFIT5 | IFI16 | interferon induced protein | Activation of innate immune response | - | - | - | ||
LRRC18, LRRTM4, LUZP2 | LRIG1 | leucine-rich repeat protein | Cytokine mediated signaling | - | - | - | ||
PROM2 | PROM1 | prominin | Cholesterol/cadherin/actinin binding | - | - | - | ||
RGS4 | RGS5 | regulator of GPCR | G-protein coupled receptor activity | - | - | - | ||
SLC10A2, SLC4A9 | SLC35F1, SLC7A7 | solute carrier family | Membrane transporter activity | - | - | - | ||
DOWN | ATP8B3 | ATP13A3 | ATPase | Membrane transporter activity | - | - | - | |
HOXB9, HOXD12 | HOXA11B | homeobox | Cell chemotaxis, transcription factor activity | - | - | - | ||
TBX4 | TBX20 | T-box family | Transcription factor activity | - | - | - | ||
Permutation tested differentially expressed genes are also overrepresented in the category of immune functions
Since our previous analysis based on the top 200 differentially expressed genes (p < 0.05) based on fold changes (largest to smallest order) may contain a number of false positives, we performed an additional analysis using just the significantly differentially expressed genes (as determined by a 5% experiment-wide permutation threshold). These results are presented in the Additional file 6: Table S6, Additional file 7: Table S7, Additional file 8: Table S8, Additional file 9: Table S9, Additional file 10: Table S10 and Additional file 11: Table S11. In summary, a few immune function categories were enriched, while the GO analysis failed to detect immune functional genes in the chicken (for multiple group comparisons see Additional file 6: Table S6 and for pairwise comparison see Additional file 7: Table S7 and Additional file 8: Table S8). In the pig, differentially expressed genes (for multiple group comparisons see Additional file 8: Table S8 and for pairwise comparison see Additional file 9: Table S9, Additional file 10: Table S10 and Additional file 11: Table S11) belonged to the immune function categories revealed by both enrichment and GO analysis. However, in both species the extent of over-representation of the immune function GO category was less when using only the genes that were significant with the permutation test, though in both cases fewer genes were used in the analyses.
Discussion
In the present experiments the pattern of gene expression changes registered in the oviduct sperm reservoirs of zoologically distant modern, fertility-selected chicken and pigs were studied 24 h after mating or in vivo SF/SP-infusion. We find that both mating and sperm-free SF/SP insemination causes gene expression changes in the primary functional sperm reservoirs of hens and sows, as detected by cDNA microarray. One caveat with this is that two different microarrays have been used for these analyses (Affymetrix and Roche), which could lead to some variation in the results, despite the raw microarray data being normalized and processed similarly to provide a valid match of the pattern of gene expression changes between species.
Mating and SF/SP-infusion modify gene expression in the oviductal sperm reservoirs in chicken and pigs
In the present study, the level of significance at an FDR adjusted P value < 0.05 excluded almost all genes, in fact all genes tested using domestic WL-chicken were excluded and only few genes in mated pigs were found to be significant. Although we are aware of the inclusion of several false positives (type I errors), we considered differentially expressed genes at a p-value of < 0.05 irrespective of FDR correction as suggestive, to compared the gene expression between commercial layer chickens and high fertility. Interestingly, the present results using WL-chickens differ with our previous study based on an Advanced Intercross Line (AIL, an intercross between Red Junglefowl and White Leghorn chickens) using the same platform, where fifteen genes were differentially expressed after mating [9]. Even more interestingly, the ancestor Red Junglefowl showed a more than 50-fold stronger differential expression [8] in response to mating or sperm-free SF-infusion, as compared to the AIL [9] and the WL here reported. Considering all these results, we assume that selection for higher fertility, at least in the chicken, might have an effect on gene expression in the oviductal sperm reservoirs after mating or artificial fertilization using a sperm-free SF-infusion. Consequently, we consider it possible that domestication and the selection for higher fertility has made the domestic hen oviduct less responsive to antigenic spermatozoa and seminal fluid. We cannot assume the same is happening in the pig, since we have not compared the modern pig with wild boar (Sus scrofa) under the same experimental conditions (mating or SP-infusion with controls) nor do we have evidence of such comparative studies being performed elsewhere.
The current results show that both mating and SF/SP-infusion are separately capable of modifying gene expression in the sperm reservoir (Fig. 2). However, irrespective of either mating or SF/SP-infusion, the number of differentially expressed genes varies; in the chicken, a large subset of genes were upregulated and comparatively a smaller subset of genes were downregulated (compare Fig. 2a with b), while in pigs, a small subset of genes were upregulated and comparatively a larger subset of genes were downregulated (compare Fig. 2c with d). Again in the chicken, a larger subset of differentially expressed genes in the UVJ were responsive to SF-infusion (476 upregulated and 387 downregulated), while a comparatively smaller subset of differentially expressed genes responded to mating (upregulated 152 and downregulated 83). In contrast, the pig UTJ responded to mating with modifications of gene expression for a larger subset of differentially expressed genes (upregulated 511 and downregulated 619), while SP-infusion only modified a comparatively small subset of differentially expressed genes (upregulated 213 and downregulated 343). The results indicate mating and/or SF/SP-infusion are able to induce gene expression changes including a certain subset of genes common to both treatments, primarily in pigs although a small number were also present in chickens (see the number of common genes shown in the Venn diagrams in Fig. 2).
The presence of spermatozoa in the sperm reservoir changed gene expression in the UVJ of the chicken [9–11] and in the oviduct of mice [7], similar to our current results. One could argue that since mating is the combination of spermatozoa and SF the subset of gene expression changes by the sperm-free SF-infusion should not differ from those differentially expressed genes modified by mating. Differences in anatomical location of the functional sperm reservoirs between the species might have influenced the dissimilar results obtained with the SF. For instance, the UVJ is quite close to the site of semen (or SF) deposition, while in the pig the UTJ is more distant from the cervix. However, any fluid placed in the cervix of pigs during artificial insemination is propelled to the UTJ within minutes, by way of contractions of the myometrium [40], an effect that is increased when seminal plasma is used [41]. Seminal fluid, which sperm are transported in in while being deposited into the female genitalia, contains a complex mixture of biological molecules, some of them (TGF-β, spermadhesins, β-defensins etc.) adsorbed to the sperm surface [19, 21, 28, 42, 43], that can be carried up to the oviduct by uterine contractions, the latter influenced by other SF-components, including hormones. SP-spermadhesins can for instance be adsorbed to the plasma membrane and transported to the UTJ [44] or all the way up to the oocyte zona pellucida [45]. However, it is still unclear whether sperm-free-SF/SP is able to reach to the oviductal sperm reservoir post-infusion. It has been shown that small- to medium-size molecules (similar to those components of the SP) suspended in buffer can pass to the oviduct of the pig, after cervical insemination [46]. Male chicken seminal fluid contains proteins identified as participating in defence and immunity processes [18, 19], also observed in our previous unpublished results. Chicken semen expresses different types of β-defensins, apparently to protect spermatozoa from microbial damage [47]. As well, TGF-β isoforms known to coat the surface of human spermatozoa [42], elicit changes in the UVJ of turkey hens [10].
To the best of our knowledge, this is the first study reporting that sperm-free-SF modifies gene expression in the oviduct sperm reservoirs of both chickens and pigs, with certain gene expression changes common to either semen or sperm-free SF deposition. Such results reinforce previous findings in cervical cells [48] and uterus [24] where components of the SF play central roles, including peptides, proteins and even microRNAs [49]. Sperm-free SP has been reported as being necessary to increase the expression of genes mainly related to cytokine synthesis in the mouse uterus [24]. A similar study found, however, low gene expression changes in the oviduct of the very same species [7]. Interestingly, our present results showed that sperm-free SF/SP could be a central player for gene expression changes related to cytokine production in the sperm reservoir (Tables 2 and 4). On the other hand, mating-induced enrichment in this GO category is either absent in chickens UVJ (Table 1) or very low in pig UTJ (Table 3). These results suggest, in agreement with Schjenken et al. [24], that there are components in the SF/SP that modulate cytokine production in the female, including genomic changes.
The local immune defence is modulated by either mating or SF-infusion
The shift, either induced by mating or by sperm-free SF-infusion, of genes belonging to the immune function category of GO biological process is statistically overrepresented when compared with the reference genome in both species (Tables 1, 2, 3 and 4). Interestingly, the insemination of sperm-free SF caused the highest overrepresentation of a larger number of immune system function categories in both species compared to mating, in either species. Within mating, however, the fold enrichment in the statistical overrepresentation was found to be highest in the chicken. Considering that mating implies that both SF/SP and spermatozoa are involved, the data suggest that the presence of spermatozoa potentially suppress the influence of components of the seminal fluid. Similar functions, albeit to a lesser extent, are also revealed by the bioinformatics analysis of the more stringent subset of differentially expressed genes significant at a 5% permutation threshold (Additional file 6: Tables S6, Additional file 7: Table S7, Additional file 8: Table S8, Additional file 9: Table S9, Additional file 10: Table S10 and Additional file 11: Table S11). Pathway analysis of the top 200 differentially expressed genes showed that most of these genes were involved in the GO category of cellular and metabolic processes (Fig. 3a-b) in both the chicken and the pig. This finding is consistent for post-mating studies in mice [7] and in a chicken AIL (Red Junglefowl x White Leghorn) [9]. The patterns (ratio between up and downregulated genes) of mating-induced differentially expressed genes in the GO term categories were similar between species with the exception of the immune system process and stimulus-responsive genes (Fig. 3a). However, the SF-infusion upregulated a larger subset of immune system process genes (7 genes in chicken and 7 genes in pigs) compared to the smaller subset of downregulated genes in this category (3 genes in chicken and 2 genes in pigs) (Fig. 3b). Immune system process genes are considered to be one of the central players in sperm survival in the oviduct sperm reservoirs. The bioinformatics investigation of our present data revealed that a large subset of differentially expressed genes are involved in the suppression of local immune defence in the sperm reservoir in the chicken after mating (Table 5). Our previous microarray study in the AIL-chicken, which has a moderate egg-laying capacity [50], showed that mating induced immune modulatory gene expression changes [9]. Das et al. 2009 [11] reported that immune modulatory TGFβ isoforms and their receptors are expressed in the UVJ of WL-hens in the presence of resident sperm. In contrast, mating-induced expression changes of immune system process genes in the UTJ of pigs were largely involved in immune activation. A microarray study in mice [7] reported that immune defence genes were also upregulated in the oviduct after mating. In all these studies, the interval between sperm deposition and the gene expression changes was restricted, covering the time spermatozoa were present in the sperm reservoir, and activation could thus be considered to play a role in the elimination of redundant spermatozoa and foreign proteins/pathogens, cleansing the internal genital tract for the descending embryos. In the chicken, where such events of internal embryo development do not exist, spermatozoa are present for weeks in the sperm reservoirs and the initial activation has to be rapidly changed to suppress the immune rejection of the foreign spermatozoa, thus protecting the sperm prior to transport to the site of fertilization. To what extent the sperm-free SF interplays with the above events remains to be explored, particularly in relation to which components signal the genomic shifts that we observed.
Chicken and pig oviduct sperm reservoirs conserve common mechanisms of pH-regulation and immune-modulation
To assess the potential for overlap in genes within a similar functionality group potentially common between chicken and pig, the top 200 differentially expressed genes (100 upregulated and 100 downregulated genes) whose expression was modified either by mating or by SF-infusion in chicken or in pig, were compared. A total of 30 genes of few functional categories were shared between chickens and pigs amongst the most differentially expressed (i.e. within the top 200) in each comparison (Table 6). The genes that were identified in the common functional categories for chickens and pigs from these top 200 differentially expressed genes play prominent functions in either species, such as pH regulation (cell membrane transporters-solute carrier family genes e.g. avian SLC16A2, SLC4A9, SLC10A2 or porcine SLC13A1, SLC35F1, SLC7A7 and ATPases genes e.g. avian ATP8B3 or porcine ATP13A3) or immune-modulation (metallopeptidases genes e.g. avian MMP27 or porcine ADAMTS3, MMP3, MMP12 and interferon induced protein related genes- e.g. avian IFIT5 or porcine IFI16), confirming previous findings in the chicken using an AIL [9] and in mice [7] oviducts.
Spermatozoa are apparently quiescent while stored in the oviduct functional sperm reservoir, their motility increasing when leaving the reservoir [51–53]. Sperm motility is highly sensitive to pH and it is rapidly affected by changes in pH levels. In domestic poultry (chickens, quails and turkeys) and mammals (cows and pigs), in vitro studies revealed that sperm motility is highest at an alkaline pH and it is possible to alter them towards quiescence if they are exposed to a low pH [54, 55]. In chickens, pH values below 7.8 inhibit sperm motility, and at this level sperm motility remains low, while raising the pH value 0.2 units and higher provides vigorous sperm motility [53]. The pig cauda epididymis has a pH value around 6.5 with quiescent spermatozoa [56]; their motility becoming activated by exposure to high pH or increasing bicarbonate levels [55, 56]. The sperm reservoirs of the sow register lower pH levels (6.7) compared to the upper tubal segments where fertilization takes place (ampullary-isthmic junction: 7.5; ampulla: 8.3 [54]) adding circumstantial evidence to the suggestions that changes in pH from acidic to alkaline would also regulate sperm transfer to the fertilization site [57]. The genes of solute carrier family and ATPases are involved in pH regulation by exchanging protons, ions and HCO3 − between the intra and the extracellular space [58–61]. Regulation of sperm motility in the oviduct sperm reservoir of either species might, therefore, be controlled through modifications of the expression of these genes of the solute carrier family and ATPases, such as the ones reported here by mating and SF/SP-infusion.
The identified genes of immune modulatory function that were common between animal classes were either upregulated or downregulated by either mating or sperm-free SF/SP. Matrix metallopeptidase genes (MMP27 in chicken, or their counterparts in pig ADAMTS3, MMP3 and MMP12) were downregulated post-mating while genes controlling interferon-induced proteins (IFIT5 in chicken, or IFI16 in pig) were upregulated after SF/SP-infusion (see Table 6). Matrix metalloproteinases (MMPs) and interferon-induced proteins were previously detected in the oviduct of chickens [62], mice [63] and cows [64]. The previous reports suggested that matrix metalloproteinases are involved in immunomodulation [65]. The MMP-27 gene is expressed in the CD163+/CD206+ M2 macrophages in the cycling human endometrium [66], MMP-3 KO-mouse reduced neutrophil influx in immune-mediated lung injury [67] and macrophage number in atherosclerotic plagues [68]. The MMP-12 KO-mouse also reduced neutrophil influx in immune-mediated lung injury [69], macrophage migration [70] and reduced active TNF-α release from macrophages [71]. Therefore, downregulation of these genes might have an immune-suppressive role in the oviductal sperm reservoir in either animal class hereby considered. However, SF-insemination upregulated the expression of IFIT5 and IFI16 that might play role in immune-activation in the sperm reservoir since these genes were reported to have potential roles in enhancing innate immune and inflammatory response [72, 73]. Interestingly, these genes (IFIT5 and IFI16) were not upregulated post-mating in the sperm reservoir, and appeared to be suppressed by the presence of spermatozoa in either species, following our bioinformatics analysis of overrepresented immune function categories. This suggests these genes help create an immune-balanced physiological environment tailored for sperm survival. However, more research is necessary to expand upon such mechanisms.
Conclusion
Chickens and pigs apparently share common functional genes that induce changes post-mating that influence mechanisms for pH-regulation. The upregulated genes are often found to be the solute carrier family genes (SLC16A2, SLC4A9, SLC35F1 and SLC35F1), whilst matrix metalloproteinases (MMP27, ADAMTS3, MMP3 and MMP12) are downregulated, indicating potentially conserved mechanisms govern fertility in these two species.
Notes
Abbreviations
- cDNA:
-
Complementary deoxyribonucleic acid
- GO:
-
Gene ontology
- RNA:
-
Ribonucleic acid
- SF:
-
Seminal fluid
- SP:
-
Seminal plasma
- TLR4:
-
Toll-like receptor 4
- UTJ:
-
Utero-tubal junction
- UVJ:
-
Utero-vaginal junction
Declarations
Acknowledgements
We would like thank Anders Delleskog, Dan Linghammar and Rebecca Katajaama for their assistance during pig surgery. We thank Mihaela-Maria Martis, BILS (Bioinformatics Infrastructure for Life Sciences) for her help with Bioinformatics.
Funding
The project has been financed by the Research Council FORMAS, Stockholm (Project number: 221-2011-512).
Availability of data and materials
The microarray data have been deposited into public repository- Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/)- that could be found using the accession numbers GSE83621 (Roche NimbleGen chicken gene expression microarray data) and GSE83622 (Affymetrix pig gene expression microarray data).
Authors’ contributions
MA was involved in the study layout, executed the experiments, collected samples, performed analyses and wrote the first draft of the manuscript under supervision of last two senior authors (DW and HRM). All authors contributed to experiment execution, handling of data and preparation of the final versions of the manuscript. DW and HRM designed and supervised the study. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
“Not applicable”
Ethics approval
Animal husbandry and experimental handling were performed in compliance with the European Community (Directive 2010/63/EU) and current Swedish legislation (SJVFS 2015:24). Throughout all experiments, animals were handled carefully and in such a way as to avoid any unnecessary stress. The experiments were approved in advance by the “Regional Committee for Ethical Approval of Animal Experiments” (Linköpings Djurförsöksetiska nämnd) in Linköping, Sweden (permit no 75-12).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
References
- Hunter RHF. Sperm transport and reservoirs in the pig oviduct in relation to the time of ovulation. J Reprod Fertil. 1981;63:109–17.View ArticlePubMedGoogle Scholar
- Tienthai P, Johannisson A, Rodriguez-Martinez H. Sperm capacitation in the porcine oviduct. Anim Reprod Sci. 2004;80:131–46.View ArticlePubMedGoogle Scholar
- Bakst MR. Role of the oviduct in maintaining sustained fertility in hens. J Anim Sci. 2011;89:1323–9.View ArticlePubMedGoogle Scholar
- Rodriguez-Martinez H, Nicander L, Viring S, Einarsson S, Larsson K. Ultrastructure of the uterotubal junction in preovulatory pigs. Anat Histol Embryol. 1990;19:16–36.View ArticlePubMedGoogle Scholar
- Mburu JN, Rodriguez-Martinez H, Einarsson S. Changes in sperm ultrastructure and localization in the porcine oviduct around ovulation. Anim Reprod Sci. 1997;47:137–48.View ArticlePubMedGoogle Scholar
- Sasanami T, Matsuzaki M, MIzushima S, Hiyama G. Sperm storage in the female reproductive tract in birds. J Reprod Dev. 2013;59:334–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Fazeli A, Affara NA, Hubank M, Holt WV. Sperm-induced modification of the oviductal gene expression profile after natural insemination in mice. Biol Reprod. 2004;71:60–5.View ArticlePubMedGoogle Scholar
- Atikuzzaman M, Hoglund A, Johnsson M, Alvarez-Rodriguez M, Mehta R, Wright D, Rodriguez-Martinez H. Spermatozoa rather than seminal fluid drives immune-modulation in the sperm storage tubuli of Red Junglefowl hens. Reprod Domest Anim (2015) 50:44–44.Google Scholar
- Atikuzzaman M, Bhai RM, Fogelholm J, Wright D, Rodriguez-Martinez H. Mating induces the expression of immune- and pH-regulatory genes in the utero-vaginal junction containing mucosal sperm-storage tubluli of hens. Reproduction. 2015;150(6):473–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Das SC, Isobe N, Nishibori M, Yoshimura Y. Expression of transforming growth factor-β isoforms and their receptors in utero-vaginal junction of hen oviduct in presence or absence of resident sperm with reference to sperm storage. Reproduction. 2006;132:781–90.View ArticlePubMedGoogle Scholar
- Das SC, Isobe N, Yoshimura Y. Changes in the expression of interleukin-1β and lipopolysaccharide-induced TNF factor in the oviduct of laying hens in response to artificial insemination. Reproduction. 2009;137:527–36.View ArticlePubMedGoogle Scholar
- Huang A, Isobe N, Obitsu T, Yoshimura Y. Expression of lipases and lipid receptors in sperm storage tubules and possible role of fatty acids in sperm survival in the hen oviduct. Theriogenology. 2016;85:1334–42.View ArticlePubMedGoogle Scholar
- Georgiou AS, Snijders APL, Sostaric E, Aflatoonian R, Vazquez JL, Vazquez JM, Roca J, Martinez EA, Wright PC, Fazeli A. Modulation of the oviductal environment by gametes. J Proteome Res. 2007;6:4656–66.View ArticlePubMedGoogle Scholar
- Almiñana C, Caballero I, Heath PR, Maleki-Dizaji S, Parrilla I, Cuello C, Gil MA, Vazquez JL, Vazquez JM, Roca J, Martinez EA. The battle of the sexes starts in the oviduct: modulation of oviductal transcriptome by X and Y-bearing spermatozoa. BMC Genomics. 2014;15:293.View ArticlePubMedPubMed CentralGoogle Scholar
- López-Úbeda R, García-Vázquez FA, Romar R, Gadea J, Muñoz M, Hunter RHF, Coy P. Oviductal transcriptome is modified after insemination during spontaneous ovulation in the sow. PLOS ONE. 2015;10(6):e0130128.View ArticlePubMedPubMed CentralGoogle Scholar
- Long EL, Sonstegard TS, Long JA, Van Tassell CP, Zuelke KA. Serial analysis of gene expression in turkey sperm storage tubules in the presence and absence of resident sperm. Biol Reprod. 2003;69:469–74.View ArticlePubMedGoogle Scholar
- Holt WV, Fazeli A. Sperm storage in the female reproductive tract. Ann Rev Anim Biosci. 2016;4:291–310.View ArticleGoogle Scholar
- Marzoni M, Castillo A, Sagona S, Citti L, Rocchiccioli S, Romboli I, Felicioli A. A proteomic approach to identify seminal plasma proteins in roosters (Gallus gallus domesticus). Anim Reprod Sci. 2013;140:216–23.View ArticlePubMedGoogle Scholar
- Labas V, Grasseau I, Cahier K, Gargaros A, Harichaux G, Teixeira-Gomes AP, Alves S, Bourin M, Blesbois E. Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen. J Proteomics. 2015;112:313–35.View ArticlePubMedGoogle Scholar
- Sasanami T, Izumi S, Sakurai N, Hirata T, Mizushima S, Matsuzaki M, Hiyama G, Yorinaga E, Yoshimura T, Ukena K, Tsutsui K. A unique mechanism of successful fertilization in a domestic bird. Sci Rep. 2015;5:7700.View ArticlePubMedPubMed CentralGoogle Scholar
- Yudin AI, Generao SE, Tollner TL, Treece CA, Overstreet JW, Cherr GN. Beta-defensin 126 on the cell surface protects sperm from immunorecognition and binding of anti-sperm antibodies. Biol Reprod. 2005;73:1243–52.View ArticlePubMedGoogle Scholar
- Robertson SA, Guerin LR, Bromfield JJ, Branson KM, Ahlström AC, Care AS. Seminal fluid drives expansion of the CD4+CD25+ T regulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol Reprod. 2009;80:1036–45.View ArticlePubMedPubMed CentralGoogle Scholar
- Druart X, Rickard JP, Mactier S, Kohnke PL, Kershaw-Young CM, Bathgate R, Gibb Z, Crossett B, Tsikis G, Labas V, Harichaux G, Grupen CG, de Graaf SP. Proteomic characterization and cross species comparison of mammalian seminal plasma. J Proteomics. 2013;91:13–22.View ArticlePubMedGoogle Scholar
- Schjenken JE, Glynn DJ, Sharkey DJ, Robertson SA. TLR4 signaling is a major mediator of the female tract response to seminal fluid in mice. Biol Reprod. 2015;93(3):68.View ArticlePubMedGoogle Scholar
- Rodriguez-Martinez H, Kvist U, Saravia F, Wallgren M, Johannisson A, Sanz L, Peña FJ, Martinez EA, Roca J, Vazquez JM, Calvete JJ. The physiological roles of the boar ejaculate. In: Rodriguez-Martinez H, Vallet JL, Ziecik AJ, editors. Control of Pig Reproduction VIII”. UK: Nottingham University Press; 2009. p. 1–21. ISBN 978-1-904761-39-6.Google Scholar
- Rodriguez-Martinez H, Saravia F, Wallgren M, Martinez EA, Sanz L, Roca J, Vazquez JM, Calvete JJ. Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow. J Reprod Immunol. 2010;84:57–65.View ArticlePubMedGoogle Scholar
- Robertson SA. Seminal plasma and male factor signaling in the female reproductive tract. Cell Tissue Res. 2005;322:43–52.View ArticlePubMedGoogle Scholar
- Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez S. Bovine seminal plasma proteins PDC-109, BSP-A3, and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biol Reprod. 2006;75:501–7.View ArticlePubMedGoogle Scholar
- Rodriguez-Martinez H, Kvist U, Ernerudh J, Sanz L, Calvete JJ. Seminal plasma protein: what role do they play? Am J Reprod Immunol. 2011;66 Suppl 1:11–22.View ArticlePubMedGoogle Scholar
- Juyena NS, Stelletta C. Seminal plasma: An essential attribute to spermatozoa. J Androl. 2012;33:536–51.View ArticlePubMedGoogle Scholar
- Atikuzzaman M, Sanz L, Alvarez-Rodriguez M, Rubér M, Wright D, Calvete JJ, Rodriguez-Martinez H. Selection for higher fertility reflects in the seminal fluid proteome of modern domestic chicken. Comp Biochem Physiol Part D Genomics Proteomics. 2016;21:27–40.View ArticlePubMedGoogle Scholar
- Schütz KE, Jensen P. Effects of resource allocation on behavioural strategies: a coparison of Red Junglefowl (Gallus gallus) and two domesticated breeds of poultry. Ethology. 2001;107:753–65.View ArticleGoogle Scholar
- Johnsson M, Gustafson I, Rubin C-J, Sahlqvist A-S, Johnsson KB, Kerje S, Ekwall O, Kämpe O, Andersson L, Lensen P, et al. A sexual ornament in chickens is affected by pleiotropic alleles at HAO1 and BMP2 selected during domestication. PLoS Genetics. 2012;8:e1002914.View ArticlePubMedPubMed CentralGoogle Scholar
- Bakst MR. Structure of the avian oviduct with emphasis on sperm storage in poultry. J Exp Zool. 1998;282:618–26.View ArticlePubMedGoogle Scholar
- Simonsson, A. Näringsrekommendationer och fodermedelstabeller till svin (Nutrient and metabolizable energy recommendations for swine, in Swedish). Swedish University of Agricultural Sciences, SLU Info. Uppsala: Rapporter, Husdjur; 1994. pp. 75.Google Scholar
- Benjamini Y, Hochberg Y. Controlling the false discovery rate- a practical and powerful approach to multiple testing. J Roy Stat Soc. 1995;57(1):289–300.Google Scholar
- Mi H, Muruganujan A, Casagrande JT, Thomas PD. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 2013;8:1551–66.View ArticlePubMedGoogle Scholar
- Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.View ArticlePubMedPubMed CentralGoogle Scholar
- The UniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 2015;43:D204–12.View ArticleGoogle Scholar
- Rodriguez-Martinez H, Saravia F, Wallgren M, Tienthai P, Johannisson A, Vázquez JM, Martínez E, Roca J, Sanz L, Calvete JJ. Boar spermatozoa in the oviduct. Theriogenology. 2005;63:514–35.View ArticlePubMedGoogle Scholar
- Viring S, Einarsson S. Effect of boar seminal plasma on uterine and oviductal motility in oestrous gilts. Acta Vet Scand. 1980;21(4):607–16.PubMedGoogle Scholar
- Chu TM, Nocera MA, Flanders KC, Kawinski E. Localization of seminal plasma transforming growth factor-beta 1 on human spermatozoa: an immunocytochemical study. Fertil Steril. 1996;66:327–30.View ArticlePubMedGoogle Scholar
- Yudin AI, Tollner TL, Treece CA, Kays R, Chen GN, Overstreet JW, Bevins CL. Beta-defensin 22 is a major component of the mouse sperm glycocalyx. Reproduction. 2008;136:753–65.View ArticlePubMedPubMed CentralGoogle Scholar
- Calvete JJ, Ensslin M, Mburu J, Iborra A, Martínez P, Adermann K, Waberski D, Sanz L, Töpfer-Petersen E, Weitze KF, Einarsson S, Rodríguez-Martínez H. Monoclonal antibodies against boar sperm zona pellucida-binding protein AWN-I. Characterization of a continuous antigenic determinant and iimunolocalization of AWN epitopes in inseminated sows. Biol Reprod. 1977;57:735–42.View ArticleGoogle Scholar
- Rodríguez-Martínez H, Iborra A, Martínez P, Calvete JJ. Immunoelectronmicroscopic imaging of spermadhesin AWN epitopes on boar spermatozoa bound in vivo to the zona pellucida. Reprod Fertil Dev. 1998;10:491–7.View ArticlePubMedGoogle Scholar
- Einarsson S, Jones B, Larsson K, Viring S. Distribution of small- and medium-sized molecules within the genital tract of artificially inseminated gilts. J Reprod Fertil. 1980;59(2):453–7.View ArticlePubMedGoogle Scholar
- Das SC, Isobe N, Yoshimura Y. Expression of toll-like receptors and avian β-defensins and their changes in response to bacterial components in chicken sperm. Poultry Sci. 2011;90:417–25.View ArticleGoogle Scholar
- Sharkey DJ, Schjenken JE, Mottershead DG, Robertson SA. Seminal fluid factors regulate activing A follistatin synthesis in female cervical epithelial cells. Mol Cell Endocrinol. 2015;417:178–90.View ArticlePubMedGoogle Scholar
- Schjenken JE, Zhang B, Chan HY, Sharkey DJ, Fullston T, Robertson SA. miRNA regulation of immune tolerance in early pregnancy. Am J Reprod Immunol. 2016;75(3):272–80.View ArticlePubMedGoogle Scholar
- Schütz K, Kerje S, Carlborg Ö, Jacobsson L, Andersson L, Jensen P. QTL analysis of a Red Junglefowl X White leghorn intercross reveals trade-off in resource allocation between behavior and production traits. Behav Genet. 2002;32:423–33.View ArticlePubMedGoogle Scholar
- Bakst MR. Zinc reduces turkey sperm oxygen uptake in vitro. Poult Sci. 1985;64:564–6.View ArticlePubMedGoogle Scholar
- Bakst MR, Richards MP. Concentrations of selected cations in turkey serum and oviductal mucosae. Poult Sci. 1985;64:555–63.View ArticlePubMedGoogle Scholar
- Suarez SS. Regulation of sperm storage and movement in the mammalian oviduct. Int J Dev Biol. 2008;52:455–62.View ArticlePubMedGoogle Scholar
- Holm L, Wishart GJ. The effect of pH on the motility of spermatozoa from chicken, turkey and quail. Aim Reprod Sci. 1998;54:45–54.View ArticleGoogle Scholar
- Rodriguez-Martinez H. Role of the oviduct in sperm capacitation. Theriogenology. 2007;68(Suppl):S138–46.View ArticlePubMedGoogle Scholar
- Rodriguez-Marinez H, Ekstedt E, Einarsson S. Acidification of the epididymal fluid in the boar. Int J Androl. 1990;13:238–43.View ArticleGoogle Scholar
- Holm L, Ridderstråle Y, Knutsson P-G. Localization of carbonic anhydrase in the sperm storing regions of the domestic hen oviduct. Acta Anatomica. 1996;156:253–60.View ArticlePubMedGoogle Scholar
- Bublitz M, Preben M, Nissen P. P-type ATPases at a glance. J Cell Sci. 2011;124:2515–9.View ArticlePubMedGoogle Scholar
- Palmgren MG, Nissen P. P-type ATPases. Ann Rev. 2011;40:243–66.Google Scholar
- Casey JR, Grinstein S, Orlowski J. Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol. 2010;11(1):50–61.View ArticlePubMedGoogle Scholar
- Liu Y, Wang D-K, Chen L-M. The physiology of bicarbonate transporters in mammalian reproduction. Biol Reprod. 2012;86(4):99. 1–13.PubMedGoogle Scholar
- Lesniak-Walentyn A, Hrabia A. Expression and localization of matrix metalloproteinases 8MMP-2, −7, −9) and their tissue inhibitors (TIMP-2, −3) in the chicken oviduct during maturation. Cell Tissue Res. 2016;364:185–97.View ArticlePubMedGoogle Scholar
- Ramsey KH, Sigar IM, Schripsema JH, Shaba N, Cohoon KP. Expression of matrix metalloproteinases subsequent to urogenital Chlamydia muridarum infection of mice. Infect Immun. 2005;73:6962–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Gabler C, Odau S, Muller K, Schon J, Bondzio A, Einspanier R. Exploring cumulus-oocyte-complex-oviductal cell interactions: gene profiling in the bovine oviduct. J Physiol Pharmacol. 2008;56 Suppl 9:29–42.Google Scholar
- Parks WC, Wilson CL, López-Boado YS. Matrix metalloproteinases as modulators of inflammation and immunity. Nat Rev Immunol. 2004;4(8):617–29.View ArticlePubMedGoogle Scholar
- Cominelli A, Chevronnay HPG, Lemoine P, Courtoy PJ, Marbaix E, Henriet P. Matrix metalloproteinase-27 is expressed in CD163+/CD206+ M2 macrophages in the cycling human endometrium and in superficial endometriotic lessions. Mol Human Reprod. 2014;20(8):767–75.View ArticleGoogle Scholar
- Warner RL, Beltran L, Younkin EM, Lewis CS, Weiss SJ, Varani J, Johnson KJ. Role of stromelysin 1 and gelatinase B in experimental acute lung injury. Am J Respir Cell Mol Biol. 2001;24(5):537–44.View ArticlePubMedGoogle Scholar
- Silence J, Lupu F, Collen D, Lijnen HR. Persistence of atherosclerotic plaque but reduced aneurysm formation in mice with stromelysin-1 (MMP-3) gene inactivation. Arterioscler Thromb Vasc Biol. 2001;21(9):1440–5.View ArticlePubMedGoogle Scholar
- Warner RL, Lewis CS, Beltran L, Younkin EM, Varani J, Johnson KJ. The role of metalloelastase in immune complex-induced acute lung injury. Am J Pathol. 2001;158(6):2139–44.View ArticlePubMedPubMed CentralGoogle Scholar
- Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD. Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci. 1996;93(9):3942–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Churg A, Wang RD, Tai H, Wang X, Xie C, Dai J, Shapiro SD, Wright JL. Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-alpha release. Am J Respir Crit Care Med. 2003;167(8):1083–9.View ArticlePubMedGoogle Scholar
- Baggetta R, Andrea MD, Gariano GR, Mondini M, Rittà M, Caposio P, Cappello P, Giovarelli M, Gariglio M, Landolfo S. The interferon-inducible gene IFI16 secretome of endothelial cells drives the early steps of the inflammatory response. Eur J Immunol. 2010;40:2182–9.View ArticlePubMedGoogle Scholar
- Zhang B, Liu X, Chen W, Chen L. IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways. Acta Biochim Biophys Sin. 2013;45:867–74.View ArticlePubMedGoogle Scholar
