| 1. |
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| 2. |
- Barranco, Isabel, et al.
(författare)
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Seminal Plasma Modulates miRNA Expression by Sow Genital Tract Lining Explants
- 2020
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 10:6
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Tidskriftsartikel (refereegranskat)abstract
- The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.
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| 3. |
- Cambra, Josep M., et al.
(författare)
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Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation
- 2020
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media S.A.. - 2297-1769. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Despite its advantages for pig breeding, embryo transfer (ET) has a major handicap: high embryo mortality during the pre- and implantation period, probably caused by divergent phenomena of tolerance between the immunologically unrelated (i.e., allogeneic) embryos and the recipient sow. Thus, to reach a similar maternal tolerance as in conventional breeding by artificial insemination (AI) would be the key to ET-success. For this reason, we studied the expression of the leukemia inhibitory factor (LIF) cytokine and its receptor in the pig endometrium during the implantation period (days 18 and 24) in sows subjected to ET (AL group) vs. post-cervical-AI controls (Hemi-AL group). Quantification of expression was performed at both mRNA (rt-qPCR) and protein (WB) levels. The expression of endometrial LIF on day 24 was considerably lower in ET than in AI pregnancies. Correlations between endometrial mRNA levels of LIF and LIF-R showed that, contrary to early AI-pregnancies, ET-pregnancies lack an inverse relation between cytokine and receptor levels. In conclusion, ET-pregnancies lack sufficient endometrial levels of LIF to develop adequate immunotolerance mechanisms to prevent the rejection of allogeneic ET-embryos.
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| 4. |
- Cuello, Cristina, et al.
(författare)
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Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:3
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Tidskriftsartikel (refereegranskat)abstract
- This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of +/- 1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFG beta, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.
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| 5. |
- Cuello, Cristina, et al.
(författare)
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Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae
- 2021
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70-75%), the pregnancy loss is 5-15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip (R) Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of +/- 1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 +/- 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.
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| 6. |
- Gonzalez-Plaza, Alejandro, et al.
(författare)
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Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes
- 2022
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Ingår i: Reproduction in domestic animals. - : Wiley. - 0936-6768 .- 1439-0531. ; 57:S5, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10(-9) M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10(-9) M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2 ,7 -dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10(-9) M melatonin during in vitro maturation had no effect on these parameters.
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| 7. |
- Gonzalez-Plaza, Alejandro, et al.
(författare)
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The Open Cryotop System Is Effective for the Simultaneous Vitrification of a Large Number of Porcine Embryos at Different Developmental Stages
- 2022
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The Superfine Open Pulled Straw (SOPS) system is the most commonly used method for vitrification of pig embryos. However, this system only allows the vitrification of four to seven embryos per straw. In this study, we investigated the effectiveness of the open (OC) and closed (CC) Cryotop (R) systems to simultaneously vitrify a larger number of porcine embryos. Morulae, early blastocysts and full blastocysts were vitrified with the open Cryotop (R) (n = 250; 20 embryos per device) system, the closed Cryotop (R) (n = 158; 20 embryos per device) system and the traditional superfine open pulled straw (SOPS; n = 241; 4-7 embryos per straw) method. Fresh embryos from each developmental stage constituted the control group (n = 132). Data expressed as percentages were compared with the Fishers exact test. The Kruskal-Wallis test was used to analyze the effect of the different vitrification systems on the embryo quality parameters and two-by-two comparisons were accomplished with the Mann-Whitney U test. Differences were considered statistically significant when p < 0.05. Vitrified and control embryos were incubated for 24 h and examined for viability and quality. At the warming step, the embryo recovery rate for the CC system was 51%, while all embryos were recovered when using OC and SOPS. There were no differences between the vitrification and control groups in the postwarming viability of full blastocysts. In contrast, morulae and early blastocysts that were vitrified-warmed with the SOPS system had lower viability (p < 0.01) compared to those from the OC, CC and control groups. The embryonic viability was similar between the OC and control groups, regardless of the developmental stage considered. Moreover, the embryos from the OC group had comparable total cell number and cells from the inner cell mass and apoptotic index than the controls. In conclusion, the OC system is suitable for the simultaneous vitrification of 20 porcine embryos at different developmental stages and provides comparable viability and quality results to fresh embryos subjected to 24 h of in vitro culture.
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| 8. |
- Gonzalez-Ramiro, Henar, et al.
(författare)
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Combined synchronization and superovulation treatments negatively impact embryo viability possibly by the downregulation of WNT/beta-catenin and Notch signaling genes in the porcine endometrium
- 2022
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Ingår i: Journal of Animal Science. - : Oxford University Press. - 0021-8812 .- 1525-3163. ; 100:11
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Tidskriftsartikel (refereegranskat)abstract
- The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes >= 2 or <= 2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 +/- 10.2 and 38.3 +/- 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 +/- 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% +/- 15.2%) compared to SC and NC groups (93.8 +/- 7.6% and 91.8 +/- 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/beta-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation of beta-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk. Lay Summary Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/beta-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.
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| 9. |
- Luque, Ana, et al.
(författare)
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Noncanonical immunomodulatory activity of complement regulator C4BP(β-) limits the development of lupus nephritis
- 2020
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538. ; 97:3, s. 551-566
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Tidskriftsartikel (refereegranskat)abstract
- Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(β-). We show here that C4BP(β-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(β-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(β-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(β-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(β-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(β-) could be considered for further clinical development in patients with systemic lupus erythematosus.
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| 10. |
- Martinez, Emilio A., et al.
(författare)
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Achievements and future perspectives of embryo transfer technology in pigs
- 2019
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Ingår i: Reproduction in domestic animals. - : WILEY. - 0936-6768 .- 1439-0531. ; 54
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Tidskriftsartikel (refereegranskat)abstract
- Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non-surgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non-surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production.
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