Mating induces early transcriptional response in the rat endosalpinx: the role of TNF and RA

in Reproduction
Authors:
Lidia M ZúñigaLaboratorio de Bioquímica, Departamento Biomédico, and Centre for Biotechnology and Bioengineering (CeBiB), Universidad de Antofagasta, Antofagasta, Chile

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Juan-Carlos AndradeLaboratorio de Biología de la Reproducción, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile

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Francisca Fábrega-GuerénLaboratorio de Bioquímica, Departamento Biomédico, and Centre for Biotechnology and Bioengineering (CeBiB), Universidad de Antofagasta, Antofagasta, Chile

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Pedro A OrihuelaLaboratorio de Inmunología de la Reproducción, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

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https://orcid.org/0000-0002-4238-0932
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Ethel V VelásquezDepartamento de Tecnologías Nucleares, Comisión Chilena de Energía Nuclear, Santiago, Chile

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Elena A VidalCentro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile
ANID, Programa Iniciativa Científica Milenio, Millennium Institute for Integrative Biology iBio, Santiago, Chile

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Rodrigo A GutiérrezFONDAP Centre for Genome Regulation, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
ANID, Programa Iniciativa Científica Milenio, Millennium Institute for Integrative Biology iBio, Santiago, Chile

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Patricio MoralesLaboratorio de Biología de la Reproducción, Instituto Antofagasta, Universidad de Antofagasta, Antofagasta, Chile

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Benito Gómez-SilvaLaboratorio de Bioquímica, Departamento Biomédico, and Centre for Biotechnology and Bioengineering (CeBiB), Universidad de Antofagasta, Antofagasta, Chile

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Horacio B CroxattoInstituto de Salud Pública, Facultad de Medicina, Universidad Andrés Bello, Santiago, Chile

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Correspondence should be addressed to L M Zúñiga or H B Croxatto; Email: lidia.zuniga@uantof.cl or horacio.croxatto@unab.cl
Free access

During mating, males provide not only the spermatozoa to fertilize the oocyte but also other stimuli that are essential for initiating and maintaining the reproductive programme in females. In the mammalian oviduct, mating regulates sperm storage, egg transport, fertilization, early embryonic development, and oestradiol metabolism. However, the main molecules underlying these processes are poorly understood. Using microarray analyses, we identified 58 genes that were either induced or repressed by mating in the endosalpinx at 3 h post-stimulus. RT-qPCR confirmed that mating downregulated the expression of the Oas1h and Prim1 genes and upregulated the expression of the Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 genes. The functional category ‘cell-to-cell signalling and interaction’ was over-represented in this gene list. Network modelling identified TNF and all-trans retinoic acid (RA) as upstream regulators of the mating-induced transcriptional response, which was confirmed by intraoviductal injection of TNF or RA in unmated rats. It partially mimicked the transcriptional effect of mating in the rat endosalpinx. Furthermore, mating decreased RA levels in oviductal fluid, and RA-receptor-gamma (RARG) exhibited a nuclear location in oviductal epithelium in both unmated and mated rats, indicating RA-RARG transcriptional activity. In conclusion, the early transcriptional response regulated by mating in the rat endosalpinx is mediated by TNF and RA. These signalling molecules regulate a cohort of genes involved in ‘cell-to-cell signalling and interactions’ and merit further studies to understand the specific processes activated in the endosalpinx to sustain the events that occur in the mammalian oviduct early after mating.

Abstract

During mating, males provide not only the spermatozoa to fertilize the oocyte but also other stimuli that are essential for initiating and maintaining the reproductive programme in females. In the mammalian oviduct, mating regulates sperm storage, egg transport, fertilization, early embryonic development, and oestradiol metabolism. However, the main molecules underlying these processes are poorly understood. Using microarray analyses, we identified 58 genes that were either induced or repressed by mating in the endosalpinx at 3 h post-stimulus. RT-qPCR confirmed that mating downregulated the expression of the Oas1h and Prim1 genes and upregulated the expression of the Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 genes. The functional category ‘cell-to-cell signalling and interaction’ was over-represented in this gene list. Network modelling identified TNF and all-trans retinoic acid (RA) as upstream regulators of the mating-induced transcriptional response, which was confirmed by intraoviductal injection of TNF or RA in unmated rats. It partially mimicked the transcriptional effect of mating in the rat endosalpinx. Furthermore, mating decreased RA levels in oviductal fluid, and RA-receptor-gamma (RARG) exhibited a nuclear location in oviductal epithelium in both unmated and mated rats, indicating RA-RARG transcriptional activity. In conclusion, the early transcriptional response regulated by mating in the rat endosalpinx is mediated by TNF and RA. These signalling molecules regulate a cohort of genes involved in ‘cell-to-cell signalling and interactions’ and merit further studies to understand the specific processes activated in the endosalpinx to sustain the events that occur in the mammalian oviduct early after mating.

Introduction

Mating induces physiological changes in the female reproductive tract to regulate reproductive function. Mating components independent of spermatozoa, such as sensorial stimulation and seminal plasma, modify the physiology of reproductive organs at various distances from the site of insemination. In this context, sensorial stimulation of the vaginal-cervical area induces ovulation in rabbits (Cervantes et al. 2015) and pseudopregnancy in spontaneous ovulators such as rats (Gunnet & Freeman 1983). In the same way, the seminal plasma induces an inflammatory response in the uterus (Robertson et al. 1996, Schjenken et al. 2015), which in turn induces the homing of regulatory T-lymphocytes to activate mechanisms of immune tolerance in mice (Guerin et al. 2011, Schjenken & Robertson 2014). This evidence shows that mating provides not only the spermatozoa to fertilize the oocyte but also other stimuli that are essential for initiating and maintaining the reproductive programme in females.

Previous studies have shown that mating induces a transcriptional response in the oviduct at 6 h in rodents and at 3 h in insects (Fazeli et al. 2004, Parada-Bustamante et al. 2007, Kapelnikov et al. 2008b). However, those high-throughput analyses were conducted in the whole organ, making further functional interpretation difficult. Some of the genes involved are specifically regulated by spermatozoa or seminal plasma (Fazeli et al. 2004, Bromfield et al. 2014), and some peptide components of seminal plasma target the oviduct (Ravi Ram et al. 2005). At the physiological level, it has been established that mating regulates terminal differentiation of the endosalpinx in insects (Kapelnikov et al. 2008a, Carmel et al. 2016), sperm storage and release in insects and South American camelids (Wong et al. 2008, Avila et al. 2010, Apichela et al. 2014), oviductal egg transport in rats (Forcelledo et al. 1981) and fertilization and early embryonic development in mice (Dean 2013, Bromfield et al. 2014). Some of these processes have been well characterized in insects by the groups of Mariana Wolfner and Yael Heifetz over the past 25 years, who have identified various peptides associated with sperm or included in the seminal plasma that can regulate specific functions in the oviduct and other reproductive organs. In mammals, the signals that arrive in the oviduct and their specific functions are poorly understood and are the focus of the present study.

We are interested in elucidating the cellular and molecular mechanisms by which mating regulates the physiology of the mammalian oviduct, using the rat as a model system. Previously, we reported that mating regulates oviductal egg transport (Forcelledo et al. 1981), oestradiol (E2) metabolism (Parada-Bustamante et al. 2007) and changes in the mechanism by which E2 accelerates oviductal egg transport (Orihuela et al. 2001, Parada-Bustamante et al. 2010). Regarding the effect of E2 on oviductal egg transport, we report that this mating effect is mediated by the cytokine TNF (Orostica et al. 2013). Interestingly, mating transiently increased the levels of TNF in the oviductal fluid at 3 h post-stimulus; moreover, the oviductal epithelium expresses the TNF receptors TNFRA and TNFRB on its luminal face (Orostica et al. 2013). These findings suggest that mating, through TNF, could modify the transcriptional profile in the epithelium as early as 3 h post-stimulus. Therefore, and because epithelial cells are the main cellular type constituting the endosalpinx, herein we proposed to characterize the mating-induced transcriptional response in the endosalpinx, the tissue that would be in close contact with gametes and embryos. To achieve this goal, we conducted a transcriptomic analysis of the rat endosalpinx using Affymetrix microarrays at 3 h after mating. We performed network modelling to identify the main mating-activated cellular processes in the endosalpinx and the possible upstream regulators of the mating-induced transcriptional response using the QIAGEN Ingenuity Pathway Analysis (IPA) platform.

Materials and methods

Animals

Ninety Sprague–Dawley rats, 26 males (400–600 g) and 64 females (240–280 g weight), were used in this study. The animals were kept under controlled environmental conditions (temperature 21–24°C; lights on between 07:00 and 21:00 h) and were provided rodent chow and water ad libitum. Under these controlled conditions, our animals ovulated at approximately 06:30 h of oestrus. The oestrus cycle was determined by daily vaginal smears, and all females were used after showing two consecutive 4-day cycles. Adult fertile males were maintained as two per cage and females as six per cage until they were used. The cages were 650 × 500 × 215 mm (L × W × H).

Mating and tissue collection

One female in proestrus was individually isolated (unmated rats) or caged with two fertile males at 22:00 h of proestrus. Thirty minutes after being caged with the males, the presence of spermatozoa in a vaginal smear or a vaginal plug was verified. Females with spermatozoa or a vaginal plug were designated mated rats and remained caged with the males until 01:30 h of oestrus, that is, 3 h after mating was verified. Then, the unmated and mated rats were sacrificed at 01:30 h of oestrus, and the oviducts were excised and placed in an embryologic culture dish with saline (0.9% NaCl) at 4°C. The oviducts were dried in tissue paper, placed in another embryologic culture dish, and flushed with 100 µL of saline using a 37½-gauge needle under a stereomicroscope. Under our environmental and experimental conditions, at 3 h after mating, sperm would have arrived in the oviduct and ovulation would have occurred approximately 5 h later. These characteristics were verified by inspecting the oviductal flushing for the presence of spermatozoa and the absence of cumulus oophorous using a stereomicroscope. Further verification was obtained by the presence of spermatozoa using a microscope. Concomitant with the analysis of oviductal flushing, endosalpinges were obtained as follows: we placed the flushed oviducts in another embryologic culture dish filled with saline at 4°C and removed the mesothelial membrane using curved microsurgical scissors and fine forceps to compensate for the oviductal folding. Then, we placed the de-folded oviduct in another embryologic culture dish, filled it with 0.4 mL of saline and cut each oviduct into four pieces. Each piece was held with fine forceps at one side, and with another set of forceps, the piece was carefully extruded to obtain the endosalpinx. After the pieces of both oviducts were extruded, we collected the tissue with a micropipette in a 0.6 mL conical tube, centrifuged it at 500 g for 10 min and collected the supernatant in another conical tube. Finally, we added 200 µL of TRIzol reagent (Ambion, cat# 15596-018) to the tissue, homogenized it by pipetting and stored it at −80°C for further total RNA purification. It is important to mention that the presence of uterine oedema was used as an inclusion criterion of early oestrus in unmated rats.

Microarray analysis

For the global transcriptome comparison between endosalpinx obtained from unmated and mated rats, three female rats were mated to six males, and three additional females were left unmated as controls. Mating and tissue collection were performed as previously described. Therefore, six individual samples were obtained from unmated (n = 3) and mated (n = 3) rats at 3 h after mating. Total RNA of endosalpinx was purified using TRIzol reagent by pipetting 20 times, without sonication and following the manufacturer’s instructions. The purity and quantity of RNA were assayed using a GENOVA UV/Visible spectrophotometer (Jenway Scientific Instruments), and samples with an absorbance (A) ratio (A260/A280) equal to 2.0 were used to perform microarray analysis. The RNA integrity of these samples was assayed using 1% denaturing agarose gel electrophoresis and ethidium bromide staining (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). Total RNA was quantified using the following equation: RNA (µg/mL) = A260 × 40 × dilution factor (100). Sperm removal from the endosalpinx of mated rats was considered unnecessary. In rats, the number of sperm cells entering the oviduct is exceedingly small in comparison to the number present in the cornua (Blandau & Money 1944), which was determined by Blandau in 1944 as being more than 200 (bound plus free sperm cells) (Blandau & Money 1944). Moreover, the quantity of RNA isolated from rat sperm was calculated as 0.1 pg/sperm (Pessot et al. 1989). Thus, assuming 200 sperm cells were bound to epithelial cells at 3 h after mating, their contribution to total RNA would be approximately 0.02 ng. We typically obtained 10 µg of total RNA in endosalpinx from unmated rats; therefore, contaminating sperm RNA would represent 0.0002% of the total RNA isolated from the endosalpinx of mated rats.

Microarray analysis was performed at the ‘Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile‘. We used six chips to hybridize six individual samples of unmated (n = 3) and mated (n = 3) rats, one chip by sample. GeneChip® Eukaryotic Poly-A RNA controls were added to 0.3 µg of total RNA from each individual sample. The whole-transcript (WT) sense target labelling assay was performed using the GeneChip® WT cDNA Synthesis and Amplification kit (Affymetrix, cat# 900672) and GeneChip® WT Terminal Labelling kit (Affymetrix, cat# 900670). Then, the labelled sample was hybridized to the GeneChip® Rat Gene 1.0 ST Array (Affymetrix, cat# 901176) using the GeneChip® Hybridization kit (Applied Biosystems/Ambion, cat# 900720) in a Gene Chip® Hybridization Oven 645 at 45°C for 16 h. The control oligonucleotide B2 and eukaryotic hybridization controls bioB, bioC, bioD and cre were added to each individual sample before hybridization. The chips were then washed and stained using the GeneChip® Wash and Stain kit (Applied Biosystems/Ambion, cat# 900720) in a GeneChip® Fluidics Station 450 (Affymetrix). Chip scanning was performed using GeneChip® Scanner 3000 7G (Affymetrix). Quality controls were performed using Expression Console software (Affymetrix), and data were normalised using Robust Multichip Analysis (RMA). Differentially expressed genes were identified using the RankProd package (Del Carratore et al. 2016) in the R platform (R-Core-Team 2017). RankProd utilizes the so-called rank product nonparametric method to identify up- or downregulated genes between two conditions (Breitling et al. 2004). The list of differentially expressed genes was selected using a P-value cut-off = 0.001. Correction for multiple testing was performed by calculation of the percentage of false positives (PFPs) (Breitling et al. 2004). Then, the list of up- and downregulated genes was selected using a PFP cut-off = 0.1 (Supplementary Tables 1 and 2). This cut-off was used as a compromise between reducing false positives while keeping false negatives at a reasonable level, as recommended previously (Norris & Kahn 2006).

The data discussed in this study have been deposited in the NCBI GEO and are accessible under accession number GSE99380 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE99380.

Network modelling and functional characterization of transcriptional changes

Microarray data were analysed using the QIAGEN IPA platform (http://www.qiagen.com/ingenuity). This software builds network models based on published data of molecular interactions obtained for human, mouse, and rat genes, contained in the Ingenuity Knowledge Base. Typical results are presented in tables and network figures (Supplementary Fig. 2 and Supplementary Table 3); with the term molecule referring to any gene, protein, miRNA, metabolite or chemical. Relationship types between molecules are represented by different arrow shapes as showed in Supplementary Fig. 3. The QIAGEN IPA platform was designed to organize biological information to gain a high-level overview of the general biology associated with a dataset. The functional categories are ranked by P-values obtained using the right-tailed Fisher’s Exact Test. The P-value identifies significantly over-represented molecules for a given functional process. Over-represented functional processes or pathways are those that have more focus molecules than expected by chance. Two types of functional analyses can be performed in this platform, analyses for a network or for a gene list. In this study, we performed both analyses to characterize the predicted networks and the validated list of genes regulated by mating in the rat endosalpinx.

Relative expression measured by RT-qPCR

To confirm transcriptional changes detected by microarray analysis we used another group of rats, five female rats were mated to 10 males, and five additional females were left unmated as controls. Mating and tissue collection were performed as previously described. Therefore, 10 individual samples from unmated (n = 5) and mated (n = 5) rats were obtained 3 h after mating. Total RNA was purified and its quality analysed as was previously described. Before performing RT, 3 µg of total RNA was incubated with DNase I (Invitrogen, cat# 18068-015) in a 15 µL reaction for 25 min at 25°C and the reaction was stopped adding 1.5 µL of EDTA and incubation at 70°C for 15 min. cDNA was synthesized using 2 µg of total RNA free of DNA, contained in 11 µL of the previous reaction. Then, 11 µL H2O (nucleosidase free) and 2 µL 0.5 µg/µL Oligo-dT (Invitrogen cat# 18418-012) were added and incubated at 70°C for 10 min, followed by a quick chill on ice. Superscript II RT (Invitrogen, cat# 18064-014) and RNaseOUT (Invitrogen cat# 10777-019) were used for cDNA synthesis. RT master mix was composed of 8 µL 5X First-Strand buffer (250 mmol/L Tris-HCl, pH 8.3 at room temperature; 375 mmol/L KCl; 15 mmol/L MgCl2), 4 µL 100 mmol/L DTT, 2 µL dNTP mix (10 mmol/L each), 1 µL RNaseOUT (40 Units) and 1 µL Superscript II RT (200 Units). This mix was added to total RNA + Oligo-dT, and 40 µL of the total reaction was incubated at 42°C for 60 min and inactivated at 70°C for 15 min. No-RT control (NRT) samples were prepared using the remaining 5.5 µL of RNA free of DNA, in which Superscript II RT enzyme was omitted.

For RT-qPCR, we mixed 1.5 µL cDNA, 1 µL forward primer, 1 µL reverse primer, 4 µL H2O and 7.5 µL PowerUp™ SYBR® Green Master Mix (Applied Biosystems, cat#A25741) in a 15 µL total reaction. The qPCR master mix contained SYBR™ Green Dye, Dual-Lock™ DNA Polymerase, Heat-labile Uracil-DNA Glycosylase (UDG), ROX™ dye Passive Reference, dNTP blend containing dUTP/dTTP and optimized buffer components. The primers (Invitrogen or IDT) were designed using OligoPerfect™ Designer (Invitrogen) and Primer-BLAST (NCBI). The screen of amplicon specificity was performed using BLAST. The qPCR programme was as follows: one initial step at 50°C for 2 min for UDG activation and a second step at 95°C for 2 min for Dual-Lock™ DNA Polymerase activation. These steps were followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 58, 60 or 62°C for 1 min and extension at 72°C for 1 min. The qPCR programme was performed in a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using white plates (Applied Biosystems cat#4346907) sealed by its MicroAmp™ Optical adhesive film (Applied Biosystems cat#4311971). The RT-qPCR was carried out in duplicate for each sample, and the mean value of quantification cycle (Cq) was used for further analysis. The Cq was calculated using the Fit Point Method or Threshold Cycle in StepOnePlus™ Software v2.3 using default settings.

The relative expression of target genes was calculated using the comparative CT method (Livak & Schmittgen 2001). Because this method assumes similar qPCR efficiency (E) between target and reference genes, we previously corrected the Cq (CT) values obtained for both target and reference genes in each individual sample. Subsequently, we normalised the Cq values (Cqnorm, also known asΔCq or ΔCT) using two reference genes. These procedures were performed in GenEx 7.0 software (MultiD Analyses AB) using the following equations: Cq E100% = Cq E [Log10 (1 + E)/Log10 2] and Cqnorm = Cqtarget − ½ (Cq Actb + Cq Gapdh), respectively. NormFinder package in GeneEx 7.0 software validated the use of Actb and Gapdh as the best combination of reference genes for data normalisation among all genes tested by RT-qPCR in rat endosalpinx (Supplementary Table 4). To present and compare the relative expression of each target gene between unmated and mated rats, we used 2Cq transformation; this relative expression is related to reference genes but not to the calibrator (control group) like 2−ΔΔCq (Schmittgen & Livak 2008). The Mann–Whitney test was used to determine the group difference for significance (P < 0.05) using Prism 8 software (GraphPad).

RT-qPCR validation

Primer validation for qPCR amplification was conducted as follows: (1) The optimal annealing temperature was determined using four different temperatures for each pair of primers: 56, 58, 60 and 62°C. (2) The optimal primer concentration was determined using three different primer concentrations: 667, 222 and 74 nmol/L. (3) Amplicon specificity was determined by melting curve analysis after qPCR. It was performed as follows: one initial step at 95°C for 15 s, followed by a 60°C incubation for 1 min. Fluorescence values were acquired for each 0.3°C increase until 95°C was reached, with a final step at 95°C for 15 s. The melting curves of each analysed gene are presented in Supplementary Fig. 4A, B, C and D. Reactions using NRT or H2O (no template control, NTC) samples were used as negative controls. We also verified the expected length of the amplicons by using 2% agarose gel electrophoresis (Supplementary Fig. 5). (4) The E was determined following the protocol described by Schmittgen and Livak (2008). Briefly, we performed four steps of ten-fold serial dilution of the cDNA pool from unmated and mated cDNA samples, obtaining five different cDNA concentrations. Then, qPCR was performed under conditions producing the lowest Cq value for each analysed gene, and technical duplicates were obtained for each concentration. Next, we plotted the Cq value (y-axis) vs the log10 cDNA dilution: 1, 0.1, 0.01, 0.001 and 0.0001 (x-axis), and linear regression was used to calculate the slope of the line and the r squared (r2) values. The standard curves of each analysed gene are presented in Supplementary Fig. 6A and B. Finally, the E was calculated using the equation ‘E = 10−(1/slope) − 1’ as stated in the MIQE guidelines (Bustin et al. 2009). Ideally, E should be between 90– 110% and r2 > 0.98 for a qPCR assay (Taylor et al. 2010).

Quantification of retinoic acid in oviductal fluid

To quantify retinoic acid (RA) levels in oviductal fluid, 11 female rats were mated to 10 males and 11 additional females were left unmated as controls. For mating, we proceeded as was described in ‘Mating and tissue collection’ section; in this case, however, after the oviducts were flushed with 100 µL of saline each, we collected the flushing (oviductal fluid) with a micropipette in a 0.6-mL conical tube. A total of 200 µL of oviductal fluid was collected, cleared by centrifugation at 10.000 g for 10 min and the supernatant stored at -80°C for further analysis. To determine the levels of RA in oviductal fluid, we used a mouse RA ELISA kit (MyBioSource, cat# MBS706971). Twenty-two individual samples from unmated (n = 11) and mated (n = 11) rats were obtained at 3 h after mating. The samples were concentrated from 200 to 60 µL using Microcon®-10 centrifugal filters (Merck Millipore Ltd., cat# MRCPRT010) before being assayed. For each sample, a volume of 50 µL was added to the microtiter well, which were precoated with an antibody specific to RA; immediately, 50 µL of horseradish peroxidase (HRP)-conjugated RA was added. This kit is based on a competitive inhibition enzyme immunoassay; therefore, we dispensed alternate samples from unmated and mated rats into the microtiter well to decrease variability due to incubation time. Then, samples plus conjugate were incubated at 37°C for 40 min. Next, the samples were discarded and the wells washed four times using the wash buffer, followed by the addition of 90 µL of TMB substrate to each well and incubation at 37°C for 20 min. Colour development was stopped with 50 µL of stop solution. Finally, the optical density was measured at 450 nm with wavelength correction option at 570 nm using a microplate reader Infinite® F50 (TECAN Austria GmbH). The standard curve was prepared following the manufacturers instructions and nonlinear regression was calculated for the data using Curve Expert Professional software (Hyams Development). The logarithmic power was one of the best fit curves, and its equation was y = a/(1 + (x/b)c); a = 20,568; b = 0.000125; and c = 0.7142. The total protein concentration in oviductal fluid samples was quantified using the Bradford method (Bio-Rad Protein Assay, cat# 500-0006) and BSA (EMD Millipore Corp., cat#12659-25GM) was used as a protein standard. The quantity of RA in oviductal fluid was normalised by total protein. The Mann–Whitney test was used to determine the group difference for significance (P < 0.05) using Prism 8 software.

Immunofluorescence of RA-receptor gamma in oviductal tissue sections

To determine whether oviductal epithelium can sense RA in oviductal fluid, three female rats were mated to six males, and three additional females were left unmated as controls. Because this experiment was done concomitant with the precedent experiment, we used the same males. For mating, we proceeded as described in the ‘Mating and tissue collection’ section; in this case, however, the oviducts were not flushed and the complete organs were immediately fixed in cold 4% paraformaldehyde (w/v) in buffer phosphate (0.1 mol/L NaH2PO4 + 0.1 mol/L Na2HPO4, pH 7.2) at 4°C for 1.5 h. After that, the oviducts were dehydrated at 4°C by sequential 30 min incubations in 5, 10, 15, 20 and 30% sucrose (w/v) and embedded in optimal cutting temperature (O.C.T., Tissue-Tek, Cat# 4583) before being frozen in liquid N2 and stored at −20°C until cutting. Six individual samples from unmated (n = 3) and mated (n = 3) rats were obtained at 3 h after mating. Oviduct sections (0.7 µm) were obtained using a Leica CM1850 cryostat (Leica Microsystems) and mounted on positively charged slides using SuperFrost® Plus (Thermo Scientific; cat# 4951PLUS4). Then, the tissue sections were permeabilized in cold acetone for 5 min and incubated in blocking buffer (PBS pH 7.0 + 0.2% (v/v) Triton X-100 + 2% (w/v) BSA) for 1 h at room temperature. Subsequently, the samples were incubated with anti-RA receptor gamma antibody (RARG; Abcam, cat# ab191368) at a 1:200 dilution in blocking buffer overnight at 4°C. The tissue sections were then washed three times in PBS-Triton (PBS pH 7.0 + 0.2% (v/v) Triton X-100) for 5 min each at room temperature, followed by incubation with anti-rabbit IgG Alexa Fluor® 594 antibody (Life Technologies, cat# A21442) for 1 h at room temperature. Then, the tissue sections were washed three times with PBS-Triton and incubated for 10 min in a drop of 0.05 mol/L Hoechst 33258 (Sigma, Cat# 14530), washed in distilled water and left to dry. Finally, each tissue section was covered with a drop of Fluoromount-G (Electron Microscopy Science, cat# 17984-25) and a coverslip. In the negative controls, incubation with anti-RA receptor gamma antibody was omitted. Slides were imaged using a Leica TCS SP8 confocal spectral microscope (Leica Microsystems).

Intraoviductal injection of drugs to identify upstream regulators of mating-regulated genes in endosalpinx

To identify transcriptional targets of RA and TNF among mating regulated genes in endosalpinx, we used 20 female rats, which were randomly distributed in four different groups. Each group of rats received an intraoviductal injection of 5 µL of saline, 5 µL of 0.44 nmol/L recombinant rat TNF (BD Pharmigen, cat#555109), 5 µL of 10 nmol/L RA (CAYMAN Chemical, cat# CAY-11017) or 5 µL of 10 nmol/L AGN193109, an antagonist of RARs (Santa Cruz Biotechnology, cat# sc-210768) at 22:00 h of proestrus. RA and AGN193109 were dissolved in DMSO and diluted in saline, while TNF was dissolved and diluted in saline. Twenty individual samples from unmated rats injected with saline (n = 5), TNF (n = 5), RA (n = 5) or AGN (n = 5) were obtained 3 h after the procedure. The TNF dose used in this study mimicked the mating effect on E2 acceleration of oviductal egg transport in the rat (Orostica et al. 2013). The dose of RA was 3.3-fold the total quantity of RA in the oviductal fluid of unmated rats (0.68 pg/µg total protein); considering 6.7 µg (average) of total protein in the oviductal fluid of one oviduct, we calculated 4.556 pg RA/oviduct. Therefore, we used 15 pg RA contained in 5 µL of saline (3 pg/µL or 10 nmol/L). The dose of RAR antagonist (AGN193109) was equivalent to the RA dose.

Surgical intervention was conducted as follows: unmated rats were anesthetized with 75 mg/kg of ketamine + 5 mg/kg of xylazine by intraperitoneal injection. The oviducts and ovaries were exposed through flank incisions, and the blood vessels in the peri-ovarian sac were cauterized using a Small Vessel Cauterizer kit (Fine Science Tools, cat# 18000-00) before the sac was cut open to expose the fimbria. Drugs or their vehicle (saline) were injected through the fimbria into the oviductal lumen using a Hamilton syringe (Hamilton Company). All procedures were performed under an OPMI 6-SDFC surgical microscope (Zeiss). Then, the peri-ovarian sac was replaced around the ovary, the organs were returned to the peritoneal cavity, and the muscles and skin were sutured. This procedure lasted 30 min and endosalpinges were obtained 3 h later and stored at −80°C in TRIzol reagent. Total RNA, RT-qPCR and data analyses were performed as previously described. Relative expression of the genes with confirmed changes by RT-qPCR were analysed in this experiment. The Kruskal–Wallis test was used for multiple comparisons, and significant differences were followed by the uncorrected Dunn test to determine group differences for significance comparing the TNF, RA or AGN group against the saline group (control group) in Prism 8 software.

We confirm that all experiments described in this section were performed in accordance with the guidelines and regulations revised and approved by our institutions and the Scientific and Bioethics Committees of ‘Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT)’, project ANID/CONICYT FONDECYT Iniciación 11121491.

Results

Network modelling and confirmation of transcriptional changes

Mating induced transcriptional changes in the rat endosalpinx at 3 h post-stimulus. In total, 58 genes were either induced or repressed in endosalpinx because of mating (Supplemental Tables 1 and 2). Given that the QIAGEN IPA platform uses information extracted from the scientific literature to establish functional relationships between molecules, the software selected only 24 well-characterized genes from our list (Table 1) and identified five different networks (Supplementary Table 3). The network with the highest score contained 35 molecules, including 10 genes from our list: Angiopoietin like 3 (Angptl3), Chondroadherin (Chad), Carbohydrate sulfotransferase 10 (Chst10), Family with sequence similarity 111 member A (Fam111a), 2′-5′Oligoadenylate synthetase 1E (Oas1e), DNA primase subunit 1 (Prim1), Ring finger protein 135 (Rnf135), Solute carrier family 6 member 14 (Slc6a14), Solute carrier family 26 member 4 (Slc26a4) and Secretory leukocyte peptidase inhibitor (Slpil2) (Supplementary Fig. 2A, shaded shapes). The second network also included 35 molecules, including six genes from our list: Aly/REF export factor (Alyref), Carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1), Protein phosphatase 2C-like domain containing 1 (Pp2d1), Solute carrier family 5 member 3 (Slc5a3), Small nuclear ribonucleoprotein D1 polypeptide (Snrpd1) and Uroplakin 3B like 1 (Upk3bl) (Supplementary Fig. 2B, shaded shapes). Therefore, we focused on the genes in these two top networks for further confirmation of the observed transcriptional changes using RT-qPCR. The primer sequences, qPCR conditions and product specificity are listed in Table 2. After data analysis, we found that mating downregulated the expression of the genes Oas1h and Prim1 and upregulated the expression of the genes Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 in endosalpinx (Fig. 1). Furthermore, mating did not change the expression of the genes Alyref, Rnf135, Slc6a14 and Snrpd1 when assayed by RT-qPCR, suggesting that they were false positives detected by microarray (Fig. 1). We could not assess the expression of Fam111a, Pp2d1, Slpil2 and Upk3bl transcripts via RT-qPCR because these genes were only marginally detected with the primers used (Supplementary Table 5). Additionally, in the case of Angptl3, we detected two bands even with two different pair of primers assayed (Supplementary Table 5). Therefore, we selected only the RT-qPCR confirmed genes for further analyses.

Figure 1
Figure 1

Validation of microarray data using RT-qPCR. Samples were obtained from unmated and mated rats at 3 h after stimulus. The scatter plot for each target gene represents the normalised individual data points, 2-ΔCq transformation, obtained by rat in the unmated and mated groups. Actb and Gapdh were used as reference genes for data normalisation. Lines in each scatter plot represent the median of the corresponding data set. The Mann–Whitney test was used to determine the group difference for significance (P < 0.05). **P < 0.01 indicates significant differences. Biological replicates, n = 5 for each group.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

Table 1

List of 24 mating-regulated genes in rat endosalpinx identified by microarray analysis.

Gene name Affymetrix ID RP/Rsum FC PFP
Alyref 10749681 234.5 −1.8 0.0729
Angptl3 10870110 225.3 0.6 0.0984
Ceacam1 10719847 342.6 0.6 0.0948
Chad 10737513 323.2 0.6 0.0994
Chst10 10927603 134.7 0.5 0.0445
Dsel 10763421 342.1 0.6 0.0978
Fam111a 10714106 279.5 0.6 0.0954
Fndc7 10826117 147.0 0.5 0.0480
Gpa33 10765356 215.8 0.6 0.0988
Lilra6 10703710 245.2 0.6 0.0793
LOC100909409 10806198 232.1 −1.7 0.0755
Nxpe2 10917087 218.4 −1.7 0.0695
Oas1e 10758785 123.7 −2.2 0.0211
Olr1298 10848118 301.8 0.6 0.1013
Pp2d1 10921251 116.6 −3.2 0.0223
Prim1 10892939 134.9 −1.9 0.0238
Rnf135 10736606 108.9 −1.9 0.0232
Slc5a3 10750282 268.4 0.6 0.0879
Slc6a14 10932089 251.6 0.6 0.0805
Slc26a4 10889541 319.3 0.7 0.1038
Slpil2 10851573 91.3 −1.9 0.0175
Snrpd1 10800140 313.8 −1.7 0.1009
Tmprss11b 10772013 333.0 0.6 0.1004
Upk3bl 10761096 162.1 −1.9 0.0348

© 2000-2016 QIAGEN. All rights reserved. Genes were selected using the QIAGEN IPA platform and used to predict functional networks.

RP, Rank product; FC, Fold change; PFP, Percentage of false positives.

Table 2

Information of the set of primers used for RT-qPCR assays according the MIQE guidelines.

Gene mRNA ID Primer Sequences (5′ to 3′) Primer Conc. (nmol/L) AT (°C) Slope Efficiency Amplicon length Melting point (°C)
Forward (Exon No.) Reverse (Exon No.)
Actb NM_031144.3 (4): TGA CGG TCA GGT CAT CAC TAT C (6): GTA ACA GTC CGC CTA GAA GCA T 667 60 −3.310 1.005 412 86.32
Alyref NM_001109602.1 (3,4): TCC CTT TGG ATG CAA GCT AC (6): ACC AAA ACT GCC AGA ACC AC 667 60 −3.881 0.810 238 83.35
Ceacam1 NM_001033860.1 (3): CGA AGT GAC CCA TTC AAC CT (5): TTT GAG AAG CAG GTC AGG GT 667 60 −3.122 1.091 368 83.80
Chad NM_019164.1 (1): GGA TAA CAC CAA CCT GGA GAA G (4): TGG TAG GGG GAA CAT CAG TAG T 667 60 −3.523 0.923 397 87.23
Chst10 NM_080397.1 (4): GGC TGG AGC TCA TCA GAA AC (6): ATC TCG TGC CTG TAC CAA GG 667 60 −2.972 1.170 400 83.65
Gapdh NM_017008.4 (4): ACC ACA GTC CAT GCC ATC AC (7): TCC ACC ACC CTG TTG CTG TA 222 60 −3.268 1.023 452 88.11
Oas1e NM_001009492.1 (5,6): GTG TCG GCA CAG AAA CAA GA (6): CAG ACA GAA GCC ACG TTT GA 667 60 −3.425 0.959 298 87.68
Prim1 NM_001008768.2 (8): TTT CAC AGT TCA CCT CAG CG (9,10): ATA GGC ACA GAA ATG CGA CC 222 60 −3.097 1.103 215 83.05
Rnf135 NM_001012010.1 (2): GAG CCT TCA AAC GCA AAG AC (4): GAT GGC CCA TAG GGA AAA CT 667 60 −2.849 1.244 313 83.35
Slc5a3 NM_053715.2 (1): TTT ATA TCC AGG AGG TGG CG (1): ACG AAT CTG ATC CTT CGT GG 667 60 −3.061 1.122 311 84.09
Slc6a14 NM_001037544.1 (1): ATG GAC AGA TTG AAG TGC CC (3): GAA GAA CAA AGG CAA GCC AG 667 62 −3.447 0.950 273 82.60
Slc26a4 NM_019214.1 (8): TGG CTT ACG CTA TTG CAG TG (10,11): AGA CTT CTG CAA GGG TTC CA 667 58 −3.229 1.040 269 86.48
Snrpd1 NM_001106163.1 (1): TCT TCC GGC CAT TCA TAC TC (3): GGT GCG TAT TCA TGC TGA CA 667 60 −3.066 1.119 207 82.46

AT, annealing temperature; conc, concentration.

Network modelling predicts that RA is an upstream regulator of the mating transcriptional response in the rat endosalpinx

To model confident relationships among genes regulated by mating in the rat endosalpinx, we performed network modelling using only the seven genes with confirmed changes by RT-qPCR. The analysis using QIAGEN IPA platform identified three different networks (Table 3). The top network comprised 35 molecules, including five genes from our new list: Ceacam1, Chad, Chst10, Slc5a3 and Slc26a4 (Fig. 2, shaded forms). More interestingly, in this network we identified a common upstream regulator of mating regulated genes in the rat endosalpinx, tretinoin, also known as RA. Network analysis did not predict TNF as another upstream regulator of mating transcriptional response as was expected. Therefore, we undertook an additional network analysis that included TNF because this cytokine is known to be increased in oviductal fluid at 3 h after mating (Orostica et al. 2013).

Figure 2
Figure 2

Network modelling predicts that RA is an upstream regulator for four mating-regulated genes in the rat endosalpinx. In this network, RA (tretinoin) is a central molecule regulating several molecules. The relationships support the idea that RA transcriptionally regulates Ceacam1, Chst10 and Slc5a3, whereas RA regulates Slc26a4 through iodide. Shaded nodes represent genes regulated by mating. Key of relationships in Supplementary Fig. 3.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

Table 3

Networks predicted by IPA involving mating-regulated genes in rat endosalpinx.

Molecules in the networks Score Focus molecules Top diseases and functions
Network 1

B3GAT1, bicarbonate, CAR ligand-CAR-Retinoic acid-RXRa, CD3, CD6, CD52, CEACAM1, CEACAM6, CEACAM8, CHAD, CHN2, CHST10, ELAVL3, ERK1/2, GLP2R, HS2ST1, HS3ST1, HS6ST1, IFI44L, iodide, KCNK5, MFNG, MIP1, MNK1/2, NDST1, NRG3, PDE6H, PTPN7, SIT1, SLC26A4, SLC5A3, SPINK4, TFF2, tretinoin, ZNF133.
13 5 Carbohydrate metabolism, small molecule biochemistry, cellular compromise.
Network 2

HRAS, NFKB (complex), Oas, Oas1h (Oas1e).
3 1 Cancer, cell cycle, cell death and survival.
Network 3

ADRA1A, ADRA1D, E2F1, epinephrine, Prim1.
3 1 Cell cycle, connective tissue development and function, hair and skin development and function.

© 2000-2016 QIAGEN. All rights reserved. Only seven genes were included in this analysis because their changes were validated by real-time RT-qPCR. These genes were involved in three different networks. Key of molecules in the networks: gene symbols from human, all letters capitalised; gene symbols from mouse and rat, first letter capitalised; other molecules, letters in roman (i.e. not italicised); and genes highlighted in bold are focus molecules.

Network modelling predicts that TNF is an upstream regulator of the mating transcriptional response in the rat endosalpinx

To identify possible TNF targets among the seven mating regulated genes in the rat endosalpinx, we performed further network modelling that included these seven genes (Ceacam1, Chad, Chst10, Oas1e, Prim1, Slc5a3 and Slc26a4) plus TNF, because we know that all of them are mating-regulated molecules in the oviduct at 3 h post-stimulus. In this case, the QIAGEN IPA platform identified only 1 network (Fig. 3 and Supplementary Table 6) comprising 24 molecules, including the eight mating-regulated molecules in the oviduct mentioned above. This unique network is associated with the ‘cell cycle’, ‘cellular growth and proliferation’ and ‘organismal functions’. More interestingly, network analysis including TNF unified the three different networks predicted in the precedent analysis (Table 3), suggesting that TNF might be the missing link among them. This unique network predicts that TNF regulates Oas1e and Prim1 expression through the activation of NFKB (Hanson et al. 2004) and E2F transcription factor 1 (E2F1) (Kalma et al. 2001), respectively, and regulates Slc26a4 through the stimulation of Interleukin 13 (IL13) (Fig. 3) (Zuo et al. 2010).

Figure 3
Figure 3

Network modelling predicts that TNF is an upstream regulator of 3 mating-regulated genes in rat endosalpinx. In this network, TNF is a central molecule regulating several molecules. The relationships support the idea that TNF transcriptionally regulates Oas1h (Oas1e) and Prim1, whereas TNF regulates Slc26a4 through IL13. Shaded nodes represent genes regulated by mating. Key of relationships in Supplementary Fig. 3.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

‘Cell-to-cell signalling and interaction’ is the top molecular and cellular function regulated by mating in the rat endosalpinx

The functional categories associated with the unique network modelled for the eight mating-regulated molecules in the oviduct are associated with the complete network comprising 24 molecules. Therefore, to outline the general biology associated only with these eight molecules, we performed a functional analysis for this gene list in the QIAGEN IPA platform. The identified molecular and cellular functions (ranked by a P-value range) are listed in Table 4. The top functional category associated with five of the eight mating-regulated molecules in the oviduct was ‘cell-to-cell signalling and interaction’. This functional category describes functions that are involved in intercellular interactions such as binding, detachment, communication, stimulation, and intercellular junction.

Table 4

IPA functional analysis for a list of eight mating regulated molecules in the rat oviduct.

Molecular and cellular functions p-value range No. of molecules Name of molecules
Cell-to-cell signalling and interaction 0.00218 – 0.00000244 5 TNF, Ceacam1, Chst10, Chad, Oas1e.
Cellular function and maintenance 0.00218 – 0.00000244 4 Ceacam1, Chst10, Slc5a3, Slc26a4.
Cell cycle 0.00174 – 0.00000455 2 Prim1, Slc26a4.
Cell death and survival 0.00218 – 0.00000585 2 Prim1, TNF.
Cellular assembly and organization 0.00174 – 0.0000611 2 Ceacam1, Chad.

© 2000-2016 QIAGEN. All rights reserved.

Mating decreases the levels of RA in the rat oviductal fluid

Because the network modelling results predicted that RA would be an upstream regulator of mating-regulated genes in the rat endosalpinx, we measured the RA levels in the oviductal fluid of unmated and mated rats at 3 h post-stimulus. The quantity of RA/total protein ranged from 0.39–1.41 to 0.35–1.13 pg/µg in unmated and mated rats, respectively. The results showed that mating decreased the RA levels in oviductal fluid from 0.68 pg/µg (0.35) to 0.51 pg/µg (0.16) (median (interquartile range)) (Fig. 4).

Figure 4
Figure 4

Mating decreases the levels of RA in oviductal fluid. RA was measured in oviductal fluids using an ELISA kit. The scatter plot represents individual data points, RA quantity as a function of total protein in oviductal fluid, obtained by rat in the unmated and mated groups. Lines in the scatter plot represent the median with interquartile range of the corresponding data set. The Mann–Whitney test was used to determine the group difference for significance (P < 0.05). * P < 0.05 indicates a significant difference. Biological replicates, n = 11 for each group.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

RARG is expressed in the rat oviduct

To determine whether epithelial cells of endosalpinx can sense RA in oviductal fluid, we evaluated the expression and subcellular location of RARG by immunofluorescence in oviductal sections of unmated and mated rats. RARG showed a nuclear location in epithelial cells under both conditions (Fig. 5), indicating that RA-RARG was transcriptionally active in both unmated and mated rats. All isthmus epithelial samples showed nuclear location of RARG; however, it was present in the cytoplasm in some ampullary epithelial cells (see right panels with 63× magnification, Fig. 5). Additionally, although RARG was expressed in the myosalpinx in both the ampullary and isthmic regions, its location was primarily cytoplasmic (Fig. 5).

Figure 5
Figure 5

Oviductal cells express RA-receptor gamma. RARG was detected in oviductal sections by immunofluorescence. Red fluorescence corresponds to RARG, and blue fluorescence corresponds to Hoechst nuclear staining. Upper panels (1° and 2° row) show sections of the ampullary region of unmated and mated rats; the second row shows the merge images from red and blue fluorescence. Lower panels (3° and 4° row) show sections of the isthmic region of unmated and mated rats; the fourth row shows the merge images from red and blue fluorescence. Left column panels are the negative controls; primary antibody was omitted. White arrows show immune cells, which could be stained by secondary antibody conjugated to Alexa Fluor 594. L, Lumen; E, Endosalpinx and M, Myosalpinx. Original magnification 20× (Barr 115.8 or 129.5 μm). Only the right column panels are at 63× (Barr 36.8 or 41.1 μm). Red arrows show the subcellular location of RARG. Images are representative of three independent experiments. Biological replicates, n = 3 for each group.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

TNF and RA regulate the expression of mating regulated genes in the rat endosalpinx

As shown in Fig. 3, TNF may be a regulatory molecule upstream of Oas1e, Prim1 and Slc26a4, while RA may be a regulatory molecule upstream of Ceacam1, Chst10, and Slc5a3, in the endosalpinx of mated rats. To validate TNF or RA as factors that regulate the expression of mating-regulated genes in the rat endosalpinx, we performed an intraoviductal injection of saline, TNF, RA or AGN193109 (RARs antagonist) at 22:00 h of proestrus in unmated rats and evaluated the relative expression of mating-regulated genes 3 h after the procedure (01:30 h). RT-qPCR analysis showed that TNF downregulated the expression of Prim1 and upregulated the expression of Chad but did not alter the expression of the genes Ceacam1, Chst10, Oas1e, Slc5a3 and Slc26a4 in endosalpinx (Fig. 6). In contrast, RA downregulated the expression of Oas1e and upregulated the expression of Ceacam1 and Chst10, but it did not alter the expression of Chad, Prim1, Slc5a3 and Slc26a4 in endosalpinx (Fig. 6). Finally, AGN193109 downregulated the expression of Slc5a3 and Slc26a4 but did not alter the expression of Ceacam1, Chad, Chst10, Oas1e and Prim1 in endosalpinx (Fig. 6).

Figure 6
Figure 6

TNF and RA regulate the expression of mating-regulated genes in the endosalpinx of unmated rats. Samples were obtained 3 h after intraoviductal injection of 5 μL of saline (0.9% w/v NaCl), TNF (0.44 nmol/L), RA (10 nmol/L) or AGN193109 (10 nmol/L, RARs antagonist) in unmated rats. The scatter plot for each target gene represents the normalised individual data points, 2−ΔCq transformation, obtained by rat in the saline (n = 5), TNF (n = 5), RA (n = 5) and AGN (n = 5) groups. Actb and Gapdh were used as reference genes for data normalisation. Lines in each scatter plot represent the median of the corresponding data set. The Kruskal–Wallis test followed by the uncorrected Dunn test were used to determine group differences for significance (P < 0.05). * P < 0.05 indicates a significant difference.

Citation: Reproduction 161, 1; 10.1530/REP-20-0190

Discussion

In this study, we report that mating induces an early transcriptional response in the rat endosalpinx at 3 h post-stimulus. This early transcriptional response suggests that the oviduct can organize a physiological response to sustain the events occurring in the oviduct early after mating. This finding also suggests that the stimulus regulating those events arrives in the oviduct in less than 3 h. This signal could be provided by the sperm, as sperm arrives in the oviduct 15 min after intercourse in rats (Blandau & Money 1944). Alternatively, it could arrive earlier via the neuronal network that connects the vagina to the oviduct (Shafik et al. 2005) in response to sensorial stimulation of the vaginal-cervical area. Indeed, in insects, mating changes the patterns of neurotransmitter release in specific regions of the oviduct (Heifetz et al. 2014). Our data support that one of the signals is TNF. This cytokine is increased in oviductal fluid 3 h after mating (Orostica et al. 2013), and an intraoviductal injection of recombinant TNF in unmated rats regulated the expression of Prim1 and Chad in the endosalpinx, partially mimicking the mating effect. Because the endosalpinx is known to not express TNF mRNA (Orostica et al. 2013), TNF cannot be released into the oviductal fluid from the endosalpinx in response to mating. TNF may arrive in the oviductal fluid in association with sperm because seminal fluid in rodents contains high levels of this cytokine (Gopichandran et al. 2006). Moreover, transcriptional changes were also reported in response to artificial insemination in a specific region of the pig oviduct, the ampullary-isthmic junction. Twenty-six differentially expressed genes were identified, eight of which were confirmed by RT-qPCR (Lopez-Ubeda et al. 2015). Although none of the genes reported in this study have been identified in the pig, TNF has been predicted to be an upstream regulator of the top network identified by QIAGEN IPA platform (Lopez-Ubeda et al. 2015), indicating that TNF may play a major role in regulating the oviductal physiology in response to sperm + seminal fluid (components of mating). However, the source of increased TNF in oviductal fluid must be elucidated to properly classify this cytokine as a stimulus provided by mating regulating transcriptional changes in the mammalian endosalpinx.

Our data also support that another signal provided by mating to the female reproductive tract is associated with the regulation of RA levels in oviductal fluid, as we observed that mating decreased the levels of this metabolite in oviductal fluid. RA is a morphogen involved in the development of many tissues and organs in vertebrates (Piersma et al. 2017). In mammals, RA regulates embryonic development and differentiation of the Müllerian duct into the female reproductive tract through RARs (Nakajima et al. 2019). In mice, the fate of oviductal stroma is determined before embryonic day 14.5 in the proximal region of the Müllerian duct, and RA-RAR activity is necessary for the development of ciliated and secretory oviductal epithelium after birth (Nakajima et al. 2016). The synthesis of RA occurs in a two-step reaction involving the oxidation of retinol (vitamin A) to retinal by retinol dehydrogenases (RDHs) and the oxidation of retinal to RA by retinaldehyde dehydrogenases (RALDH) and aldehyde dehydrogenases (ALDH) (Duester et al. 2003). The catabolism of RA is performed by Cytochrome p450 26 (CYP26), an enzyme that converts RA to polar water-soluble metabolites (Isoherranen & Zhong 2019). Retinol is obtained from dietary sources, stored in the liver, and transported in the bloodstream attached to retinol-binding protein until it arrives at its target tissue (Napoli 2017). The mouse oviduct expresses RA-synthesizing and catabolizing enzymes and their expression is compartmentalised. Aldh1 is expressed in ampullary epithelium and isthmus stroma, and Raldh2 is expressed in ampullary and isthmic epithelium as well as in the serous layer. Cyp26 is expressed in ampullary and isthmic epithelium (Vermot et al. 2000). RARs are nuclear transcription factors that belong to a family of steroid/thyroid hormone receptors and have at least three isoforms as follows: alpha (RARA), beta (RARB), and gamma (RARG) (Chambon 2004). The expression of Rara, Rarb and Rarg has been detected in the Mullerian duct in mouse embryos, and after birth, the expression of Rarb decreased and Rarg increased in the vagina and uterus during final differentiation in adulthood; however, the expression in the oviduct was not reported (Nakajima et al. 2016). We observed that RARG was expressed in the rat oviduct and showed a nuclear location in the epithelium in both unmated and mated rats, suggesting transcriptional activity of RA-RARG in the endosalpinx. Moreover, RARG was expressed by the ampullary and isthmic epithelium, indicating that the RA-RARG system is functional in both segments. The myosalpinx also expressed RARG, but in this case, it was detected outside the nucleus.

Mating decreased the levels of RA in the rat oviductal fluid; this could be explained by (1) the decreased activity of RA-synthesizing enzymes, (2) the increased activity of RA-catabolizing enzyme and/or (3) increased retention of RA by RARs in oviductal tissues to regulate transcriptional activity. Our data support the third scenario because mating does not change the nuclear location of RARG, and intraoviductal injection of RA in unmated rats regulates the expression of Chst10, Ceacam1 and Oas1h in the endosalpinx, partially mimicking the mating effect. In contrast, intraoviductal injection of an RAR-antagonist (AGN193109) in unmated rats, which could mimic the decreased level of RA in oviductal fluid, decreased the expression of Slc5a3 and Slc26a4 in endosalpinx, which is the opposite transcriptional effect of mating. Although the genes Slc5a3 and Slc26a4 were not induced by intraoviductal injection of RA, they are RA-RAR target genes in the endosalpinx during early oestrus. Thus, it is evident that to induce Slc5a3 and Slc26a4 in the endosalpinx, additional mating-regulated signals are necessary, possibly RA + TNF or IL13 based on the network analysis prediction (Fig. 3). Therefore, the mating transcriptional response in the rat endosalpinx is the result of the combined activities of at least two signals reported in the present study, TNF and RA.

Although the functions of RA in the oviduct early after mating have not been described, its crucial role in the reproductive process is widely recognized (Clagett-Dame & Knutson 2011). It has been shown that, in rats with acute nutritional vitamin A deficiency prior to mating, fertilization and embryo development often fail (80% of cases) (Evans 1928). This deleterious effect is reversed by supplying adequate doses of RA; a diet containing 2–12 µg/g is necessary to maintain normal fertilization, implantation and early embryogenesis, but embryonic death at mid-gestation often results (White et al. 1998). Thus, higher doses of RA (250 µg/g diet) are necessary by embryonic day 8.5 to support normal embryonic development and overcome mid-gestational resorption (White et al. 2000). The levels of RA in the oviduct or uterus were not measured in precedent studies, but the levels of RA necessary to restore oviductal function is concordant with the levels measured in oviductal fluid and reported in the present study (0.68 µg/g). Therefore, the finding that mating increased RA signalling in the endosalpinx and the identification of its target genes are very relevant because we could elucidate the mechanisms by which RA is necessary to sustain fertilization and early embryonic development in the mammalian oviduct.

QIAGEN IPA analysis of the eight mating-regulated molecules in the oviduct predicted a unique functional network. The probability that a limited number of molecules were included in a unique network due to chance is very low, indicating that the functions of the eight molecules reported herein strongly converged into a physiological impact on the endosalpinx early after mating. The most represented functional category among these eight molecules was ‘cell-to-cell signalling and interactions’, which includes cellular processes such as binding, detachment, communication, stimulation, and intercellular junction. The genes mediating these cellular processes in endosalpinx are Ceacam1, Chst10, Chad and Oas1e, and here we reported that they were regulated by RA and TNF. Therefore, in the following paragraphs, we describe the possible cellular processes regulated by mating through RA and TNF in the rat endosalpinx based on the functions of each identified gene.

RA increases the expression of Ceacam1 in endosalpinx, a gene encoding a glycoprotein that is expressed in nearly all epithelial tissues and mediates homotypic (through CEACAM1) and heterotypic (through other CEACAM proteins) interactions with epithelial and immune cells (Gray-Owen & Blumberg 2006). CEACAM1 is expressed by the epithelium of the Fallopian tube and is located on its luminal face (Fernandez et al. 2001), suggesting a role in mediating interactions between the oviductal epithelium and cells arriving into the oviductal lumen. Interestingly, spermatozoa express an isoform of CEACAM1 (A2) (Draberova et al. 2000) and can stablish homotypic interactions with the oviductal epithelium. Therefore, the finding that RA increases the expression of Ceacam1 in endosalpinx early after mating could be interpreted as an enhancement of the sensitivity of oviductal epithelium to detect sperm cells arriving at the oviductal lumen. Moreover, engagement of extracellular domains of epithelial CEACAMs serves as the primary stimulus for CEACAM-mediated transmembrane signalling, resulting in the enhancement of integrin-mediated adhesion to the extracellular matrix (ECM) (Muenzner et al. 2005). Thus, homotypic interactions between the epithelium and spermatozoa mediated by CEACAM1 could modify cellular adhesion of epithelial cells to the ECM in endosalpinx, which in turn could induce terminal differentiation of the oviductal epithelium since some studies have shown that enhancement of cell-ECM interactions may induce differentiation through changes in cAMP levels and the formation of functional gap junctions (El-Sabban et al. 2003). Moreover, this terminal differentiation could be related to morphological alterations in protein-synthesizing organelles (rough endoplasmic reticulum and Golgi apparatuses) and the apical specialization observed in the ampullary epithelium of the sheep oviduct at day 1.5 of pregnancy, which is viewed as the maturation of the secretory apparatus (Murray 1995).

RA also increases the expression of Chst10 in endosalpinx, a gene encoding a sulfotransferase that participates in the synthesis of the HNK-1 carbohydrate motif, which is associated with membrane lipids or proteins and mediates interactions between cells in both the immune and nervous systems (Morita et al. 2008). The roles of RA through Chst10 in epithelium have not been described, but interestingly, through the expression of Chst10, RA can suppress invasiveness in S91 murine melanoma (Zhao et al. 2009). Specifically, CHST10 acts by modifying the glycosylation state and ligand binding of α-dystroglycan to ECM (Nakagawa et al. 2012). Thus, RA through CHST10 could modify the pattern of interactions between oviductal epithelium and ECM in endosalpinx, which in turn could cooperate with the terminal differentiation of oviductal epithelium proposed previously. Additionally, CHST10 could also modify the glycosylation state of other proteins mediating interactions between epithelial cells, between epithelium and immune or nervous cells and even between epithelium and gametes arriving at the oviductal lumen early after mating. Furthermore, this sulfotransferase is located in the Golgi apparatus and is also involved in the generation of part-time proteoglycans (Hashiguchi et al. 2011). Thus, its increased expression in endosalpinx early after mating is concordant with the high activity of this organelle observed in epithelium of the sheep oviduct during early pregnancy (Murray 1995). This suggests that CHST10 would also be involved in the synthesis of sulphated proteoglycans and/or HNK-1 glycoproteins that must be secreted into the oviductal fluid early after mating. Indeed, high secretion levels of sulphated glycoproteins in rabbits occurred within 48 h of the induction of pseudopregnancy (Erickson-Lawrence et al. 1989).

TNF increases the expression of Chad in endosalpinx, a gene encoding a small leucine-rich proteoglycan that constitutes a family of secreted proteins with important roles in extracellular matrix assembly (Chen & Birk 2013). CHAD is mainly expressed by chondrocytes and regulates the formation of the collagen fibrillar network during early skeletal development (Hessle et al. 2014). The roles of CHAD in epithelium have not been described, but it has recently been proposed that CHAD acts as a tumour suppressor in hepatocellular carcinoma (Deng et al. 2017). Specifically, this proteoglycan suppresses migration and proliferation, possibly enhancing ‘focal adhesion’ and ‘ECM receptor interaction’ (Deng et al. 2017). Moreover, cellular proliferation appears to be directly inhibited by mating through TNF in endosalpinx because this cytokine downregulated the expression of Prim1. This gene encodes the catalytic subunit of DNA primase, a protein that synthesizes the RNA primer for DNA replication (Foiani et al. 1997). Therefore, its suppression is interpreted as the end of the proliferative state. Thus, mating through TNF inhibits cellular proliferation and induce ECM remodelling, perhaps with the objective of initiating terminal differentiation of the endosalpinx. In this context, it is relevant to mention that in insects mating induces terminal differentiation of the epithelium, thus increasing intercellular junctions, myofibril formation and ECM remodelling (Kapelnikov et al. 2008a,b). Similar events appear to be activated by RA and TNF in the mammalian endosalpinx to sustain the events occurring in the oviduct early after mating.

Mating may enhance other functions directly related to the survival of spermatozoa arriving at the oviductal lumen early after mating. These functions are the regulation of pH, volume, and energy metabolism in oviductal fluid; the genes involved are Slc5a3 and Slc26a4, which encode the chloride/bicarbonate and Na+/myo-inositol transporters, respectively. Ions such as bicarbonate (HCO3-) and potassium (K+) have been shown to be present in higher concentrations in oviductal fluid than in plasma (Leese et al. 2001). The concentration of bicarbonate in oviductal fluid varies with the endocrine state. For example, in monkeys, up to 35 mmol/L bicarbonate can be detected in the follicular phase (pH between 7.1–7.3), while 90 mmol/L is detected in the luteal phase (pH between 7.5–8.0) (Maas et al. 1977). No previous reports have described the expression of Slc26a4 in the oviduct of any species, but Slc26a4 expression has recently been reported to occur in mouse and rat uteri (Xie et al. 2018). SLC26A4 expression is higher during proestrus and oestrus in glandular and luminal epithelium of the uterus (Xie et al. 2018), and it is involved in the secretion of bicarbonate in uterine fluid, regulating its pH and volume during proestrus and oestrus (Xie et al. 2018). Oviductal fluid production is highest at oestrus in many species (Richardson 1981). The increase in SLC26A4 expression in the rat endosalpinx early after mating suggests that additional fluid secretion and pH regulation may be necessary. Moreover, an interesting transcriptomic analysis undertaken to identify conserved gene expression in sperm reservoirs between birds (chickens) and mammals (pigs) has reported that mating induces the expression of genes involved in pH regulation (Atikuzzaman et al. 2017). In rats, the sperm reservoir is in the isthmus region of the oviduct, and Slc26a4 expression increased by mating in the rat endosalpinx occur after the spermatozoa arrive at the oviduct. Therefore, pH regulation appears to be a conserved function regulated in the oviduct by the presence of spermatozoa, which is very interesting since bicarbonate induces sperm capacitation in vitro (Da Ros et al. 2004, Liu et al. 2012, Battistone et al. 2013). Similar to bicarbonate, the energy substrates lactate and myoinositol have also been shown to be the most abundant metabolites in bovine oviductal fluid at all stages in the oestrus cycle (Lamy et al. 2018). In rats, the myoinositol concentration has been shown to be higher in the oviduct than in the ovary or uterus (Lewin et al. 1982). These organs take up myoinositol from plasma, and its uptake increased as follows: vagina < cervix < uterus < ovary < oviduct (Lewin et al. 1982). The transporter responsible for basal myoinositol uptake in the oviduct may be SLC5A3, and its increased expression in the endosalpinx early after mating is very interesting since myoinositol has been shown to increase sperm motility and viability in vitro and in vivo (Condorelli et al. 2012, Montanino Oliva et al. 2016).

Regional expression of all these novel mating regulated genes in endosalpinx and its proposed functions must be characterized in future work since the infundibulum, ampulla and isthmus play different roles during the reproductive process that occur in the oviduct. In most mammals studied to date, the isthmic region receives the spermatozoa early after mating, and the isthmic epithelium establishes close contacts with these cells in the sperm reservoir until the time of ovulation (Hunter 2008, Suarez 2008). When ovulation occurs, cumulus oophorous are displaced into the ostium of the oviduct by the synchronized beating of cilia on the fimbriated infundibulum (Hunter 2012). The passage of the cumulus oophorous to the site of fertilization at the ampullary-isthmic junction is rapid, and fertilization and the loss of cumulus cells occur at this point (Croxatto 1996). Finally, after fertilization, embryos enter the isthmus, the transport of which towards the uterus is somewhat variable, 3–6 days; thus, early embryo development occurs at this oviductal region (Croxatto 1996, Hunter 2012). Therefore, localised expression of these seven mating regulated genes along the endosalpinx of the three oviductal regions is expected according to its proposed functions.

In conclusion, the early transcriptional response regulated by mating in the rat endosalpinx is mediated by TNF and RA. These signalling molecules regulate a cohort of genes involved in ‘cell-to-cell signalling and interactions’ and merit further studies to understand the specific processes activated in the endosalpinx to sustain the events that occur in the mammalian oviduct early after mating.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0190.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by ANID/CONICYT FONDECYT Iniciación 11121491 and ANID/CONICYT CeBiB FB-0001.

Author contribution statement

L M Z, H B C and P A O involved in study design and manuscript writing. L M Z, P A O, J C A, F A Fand E V V involved in tissue collection, surgical interventions, sample processing and RT-qPCR. L M Z, E A V and R A G contributed to microarray and data analysis. L M Z, P M and B G S contributed to data processing and table and figure preparation. All authors reviewed the manuscript.

Acknowledgements

The authors thank Mr Balbino Matheu and Mr Hernán Ubillo for their excellent animal care and B Q Mariana Ríos for instruction regarding the RNA purification and RT-qPCR. The authors also gratefully thank the Biomedical Department Director, Prof Luis Urrutia Morales, for providing the comfortable facilities and the RA ELISA KIT for the present research.

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    Figure 1

    Validation of microarray data using RT-qPCR. Samples were obtained from unmated and mated rats at 3 h after stimulus. The scatter plot for each target gene represents the normalised individual data points, 2-ΔCq transformation, obtained by rat in the unmated and mated groups. Actb and Gapdh were used as reference genes for data normalisation. Lines in each scatter plot represent the median of the corresponding data set. The Mann–Whitney test was used to determine the group difference for significance (P < 0.05). **P < 0.01 indicates significant differences. Biological replicates, n = 5 for each group.

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    Figure 2

    Network modelling predicts that RA is an upstream regulator for four mating-regulated genes in the rat endosalpinx. In this network, RA (tretinoin) is a central molecule regulating several molecules. The relationships support the idea that RA transcriptionally regulates Ceacam1, Chst10 and Slc5a3, whereas RA regulates Slc26a4 through iodide. Shaded nodes represent genes regulated by mating. Key of relationships in Supplementary Fig. 3.

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    Figure 3

    Network modelling predicts that TNF is an upstream regulator of 3 mating-regulated genes in rat endosalpinx. In this network, TNF is a central molecule regulating several molecules. The relationships support the idea that TNF transcriptionally regulates Oas1h (Oas1e) and Prim1, whereas TNF regulates Slc26a4 through IL13. Shaded nodes represent genes regulated by mating. Key of relationships in Supplementary Fig. 3.

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    Figure 4

    Mating decreases the levels of RA in oviductal fluid. RA was measured in oviductal fluids using an ELISA kit. The scatter plot represents individual data points, RA quantity as a function of total protein in oviductal fluid, obtained by rat in the unmated and mated groups. Lines in the scatter plot represent the median with interquartile range of the corresponding data set. The Mann–Whitney test was used to determine the group difference for significance (P < 0.05). * P < 0.05 indicates a significant difference. Biological replicates, n = 11 for each group.

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    Figure 5

    Oviductal cells express RA-receptor gamma. RARG was detected in oviductal sections by immunofluorescence. Red fluorescence corresponds to RARG, and blue fluorescence corresponds to Hoechst nuclear staining. Upper panels (1° and 2° row) show sections of the ampullary region of unmated and mated rats; the second row shows the merge images from red and blue fluorescence. Lower panels (3° and 4° row) show sections of the isthmic region of unmated and mated rats; the fourth row shows the merge images from red and blue fluorescence. Left column panels are the negative controls; primary antibody was omitted. White arrows show immune cells, which could be stained by secondary antibody conjugated to Alexa Fluor 594. L, Lumen; E, Endosalpinx and M, Myosalpinx. Original magnification 20× (Barr 115.8 or 129.5 μm). Only the right column panels are at 63× (Barr 36.8 or 41.1 μm). Red arrows show the subcellular location of RARG. Images are representative of three independent experiments. Biological replicates, n = 3 for each group.

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    Figure 6

    TNF and RA regulate the expression of mating-regulated genes in the endosalpinx of unmated rats. Samples were obtained 3 h after intraoviductal injection of 5 μL of saline (0.9% w/v NaCl), TNF (0.44 nmol/L), RA (10 nmol/L) or AGN193109 (10 nmol/L, RARs antagonist) in unmated rats. The scatter plot for each target gene represents the normalised individual data points, 2−ΔCq transformation, obtained by rat in the saline (n = 5), TNF (n = 5), RA (n = 5) and AGN (n = 5) groups. Actb and Gapdh were used as reference genes for data normalisation. Lines in each scatter plot represent the median of the corresponding data set. The Kruskal–Wallis test followed by the uncorrected Dunn test were used to determine group differences for significance (P < 0.05). * P < 0.05 indicates a significant difference.

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