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Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17 degrees C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen., Funding Agencies|Ministerio de Ciencia, Innovacion y Universidades; Svenska Forskningsradet Formas

Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e(-06); ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e(-05)) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). I, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; FEDER funds (EU)European Commission; Government of CataloniaAGAURAgencia de Gestio DAjuts Universitaris de Recerca Agaur (AGAUR)Generalitat de Catalunya; European Social FundEuropean Social Fund (ESF) [2018 FI_B 00236]; Government of Spain-Ministry of Education, Culture and Sports [FPU15/06029]; [PID2019-108320RJ-I00]; [IJCI-2015-24380]; [MCIN/AEI/10.13039/501100011033]

Simple Summary Glucocorticoids are steroid hormones modulating different functions in mammals, including reproduction, that act through the glucocorticoid receptor, encoded by the gene called NR3C1. Here, we describe how the expression levels of the glucocorticoid receptor change along the different compartments of the female rabbit internal reproductive tract 20 h after insemination with sperm-free seminal plasma or natural mating (whole semen) (Experiment 1) and how these levels change at 10, 24, 36, 68, and 72 h post-mating, during specific stages over time, i.e., ovulation, fertilization and the interval of early embryo development to the morula stage occurs (Experiment 2). NR3C1-upregulation was found in the infundibulum at 20 h after all treatments, especially after sperm-free seminal plasma infusion compared to mating (Experiment 1). In Experiment 2, the receptor gene expression levels increased in a spatio-temporal sequence, corresponding to the assumed location of the rabbit embryos (particularly morulae) in the oviductal various segments and timepoints (particularly 72 h), compared to down-expression at uterine regions. We conclude that NR3C1 may play a relevant role in the rabbit female reproductive tract. Rabbits are interesting as research animal models for reproduction, due to their condition of species of induced ovulation, with the release of endogenous gonadotropin-releasing hormone (GnRH) due to coitus. Glucocorticoid (GC) signaling, crucial for physiological homeostasis, is mediated through a yet unclear mechanism, by the GC receptor (NR3C1/GR). After mating, the female reproductive tract undergoes dynamic modifications, triggered by gene transcription, a pre-amble for fertilization and pregnancy. This study tested the hypothesis that when ovulation is induced, the expression of NR3C1 is influenced by sperm-free seminal plasma (SP), similarly to after mating (whole semen), along the different segments of the internal reproductive tract of female r, Funding Agencies|Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapradet, VR) [2015-05919]; Juan de la Cierva Incorporacion Postdoctoral Research Program (MICINN) [IJDC-2015-24380]; Generalitat de Catalunya, Agency for Management of University and Research Grants; European Social FoundEuropean Social Fund (ESF) [2018 FI_B 00236]; Government of Spain Ministry of Education, Culture and Sports [FPU15/06029]; Seneca Foundation Murcia (Spain)Fundacion Seneca [20780/PD/18]

Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/β hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10-72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development., Funding agencies: Research Council FORMAS, Stockholm [2017-00946, 2019-00288]; Swedish Research Council (Vetenskapsradet, VR)Swedish Research Council [2015-05919]; Juan de la Cierva Incorporacion Postdoctoral Research Program (MICINN) [IJDC-2015-24380]; [2018 FI_B 00236]