Coy Fuster, Pilar

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Coy Fuster, Pilar

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Coy Fuster, Pilar

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Universidad de Murcia. Departamento de Fisiología

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Open Access
Deciphering differences in DNA methylation and transcriptome profiles of oocytes from pigs with high and low developmental competence
(Oxford University Press, 2025-06-03) Abril Parreño, Laura; Lopes, Jordana S.; Romero Aguirregomezcorta, Jon; Galvao, Antonio; Kelsey, Gavin; Coy Fuster, Pilar; Fisiología
In vitro maturation (IVM) is a critical step in animal in vitro embryo production, yet oocytes matured in vitro often exhibit lower developmental competence than their in vivo counterparts. However, the molecular mechanisms behind this observation remain unclear. This study investigated the gene expression and DNA methylation profiles in porcine oocytes with different developmental competencies. To study these differences, we used as model oocytes from prepubertal gilts (IVM) and sows (in vivo matured) and assessed their developmental competence up to the blastocyst stage. We also examined their gene expression and DNA methylation profiles at single-cell resolution using RNA-sequencing and bisulfite-sequencing. Oocytes were obtained by aspiration of either ovarian follicles between 3-6mm diameter, and the subsequent IVM, or ovarian follicles from 8-10 mm diameter, with no need for maturation (in vivo matured oocytes). Cleavage rates (58.2±3.0 and 45.7±4.4) and blastocyst rates (31.4±3.7 and 47.5±6.6) for IVM and in vivo groups differed significantly. Using the in vivo group as a reference, IVM oocytes had 1297 down-regulated and 476 up-regulated differentially expressed genes (DEGs), with up-regulated DEGs associated with organelle organization and cell cycle processes, and down-regulated genes involved in protein synthesis and metabolomic processes. While global DNA methylation levels were similar between groups, a few differentially methylated regions were found in CpG islands, promoters, and coding regions. Our integrative analysis identified key methylated regions and genes that distinguish each group, suggesting that both donor age and maturation conditions significantly influence gene expression regulation in oocytes with different developmental competencies.
Embargo
Growth performance in pigs derived from in vitro produced embryos is enhanced compared to their artificial insemination-derived counterparts from birth to adulthood
(Elsevier, 2025-03-04) Heras García, Sonia; Soriano-Ubeda, Cristina; Quintero Moreno, Armando Arturo; Romero Aguirregomezcorta, Jon; Paris-Oller, Evelyne; Gadea Mateos, Joaquín; Romar Andrés, Raquel; Cánovas Bernabé, Sebastián; Coy Fuster, Pilar; Fisiología
Research suggests that assisted reproductive technologies may lead to adverse effects on the offspring. To date, long-term effects from fetal development through adulthood have been scarcely researched. In the present observational study, we aimed to describe growth (body weight, BW; crown-rump length, CRL; and average daily gain, ADG), and growth-related hormone levels (growth hormone, GH; insulin-like growth factor 1, IGF-1;thyroxine, T4; cortisol) of pigs derived from artificial insemination (AI) and from embryos produced in vitro under two different conditions, C-IVP, with Bovine Serum Albumin (BSA), and RF-IVP, with reproductive fluids and BSA, as protein source in culture, from birth to 5 years of age. In vitro-produced animals showed greater BW, CRL and ADG than AI. Additionally, C-IVP animals had greater BW, CRL and ADG than RF-IVP up to 6 months of age. Regarding hormones, GH concentration was greater in RF-IVP compared to AI and C-IVP. In males, IGF-1 levels of RF-IVP were greater than those of AI. Instead, T4 levels of AI males were greater than C-IVP and RFIVP. Males showed greater IGF-1 and GH than females, the latter only from 1 year old. Instead, females had greater T4 concentration than males. No differences in cortisol were observed between groups or sexes. In conclusion, this study sets reference values of growth and hormone concentrations for adult pigs that are missing in literature. Although the differences observed between the experimental groups were within physiological ranges, there was a greater growth performance in the IVP groups.
Open Access
Metabolic profile and glycemic response in fully-grown sows born using assisted reproductive technologies
(Elsevier, 2024-10-02) Cánovas Bernabé, Sebastián; Heras García, Sonia; Romero Aguirregomezcorta, Jon; Quintero Moreno, Armando Arturo; Gadea Mateos, Joaquín; Coy Fuster, Pilar; Romar Andrés, Raquel; Anatomía y Anatomía Patológica Comparada; Facultad de Veterinaria
The aim of the present work was to gain insight into the metabolism of pigs derived from assisted reproductive technologies during their adulthood. Approximately 4h after feeding, a blood sample was taken from 3.5 year old sows born by artificial insemination (AI group, n = 7) and transfer of in vitro produced embryos (IVP group, n = 11) to determine the physiological concentrations of the main biomarkers of carbohydrates (glucose and lactate), proteins (albumin, creatinine and urea) and lipids (cholesterol and triglycerides). Four weeks later, an oral glucose tolerance test (OGTT; 1.75g glucose/kg body weight) was performed after an overnight fast and 1h of water withdrawal. Blood samples were obtained prior (T = 0 min; fasting conditions) and 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min after glucose intake. At each time point, glycemia was measured immediately using glucometer test strips, and serum was collected to determine the above metabolites along with insulin and glucagon. After OGTT, the area under the curve (AUC) between sampling times and homeostasis model assessment of insulin resistance (HOMA) indices were calculated. Under physiological conditions, the concentration of metabolites studied was similar between AI and IVP sows. In both groups, fasting decreased cholesterol and increased triglycerides and urea (P < 0.001). However, creatinine and lactate were similar in both groups under physiological and fasting conditions. The expected increase in albuminemia and decrease in glycaemia after fasting was only observed in IVP sows. OGTT revealed a different glucose curve pattern (monophasic in AI and biphasic in IVP group), a lower mean concentration of cholesterol, glucose, lactate, triglycerides in IVP compared to AI pigs (P < 0.01), and a higher mean concentration of albumin, creatinine and insulin in IVP compared to AI group (P < 0.05). On the contrary, no differences were found between groups for mean serum glucagon and urea levels, nor for glucose homeostasis indices HOMA-IR and HOMA-%B. The AUC differed between groups at several time points with larger AUC for creatinine, and smaller AUC for glucose, glucagon, and triglycerides, in IVP pigs than in AI pigs at 180–210 min (P < 0.05). In conclusion, under physiological conditions the metabolic profile of fully-grown AI and IVP sows is similar and within normal ranges. Glucose challenge revealed differences in metabolic and insulin responses between groups but with normal glucose tolerance in both cases.
Open Access
Growth parameters and growth-related hormone profile in a herd of cattle up to 4 years of age derived from assisted reproductive technologies
(MDPI, 2025-02-21) Heras García, Sonia; Sena Lopes, Jordana; Quintero Moreno, Armando Arturo; Romero Aguirregomezcorta, Jon; Cánovas Bernabé, Sebastián; Romar Andrés, Raquel; Coy Fuster, Pilar; Fisiología; Veterinaria
Assisted reproductive technologies (ART) are routinely used in livestock to generate animals of high genetic value. Despite representing an outstanding accomplishment, recent studies suggest differences in health, fertility, and gestational length of in vitro-produced compared to in vivo-derived animals. Currently, there are no data available on the long-term effects of ART on growth and development. This observational study aimed to understand the relationship between growth and growth-influencing hormones in a herd of cattle derived from artificial insemination (AI) or from in vitro-produced embryos either with BSA (C-IVP) or with reproductive fluids (RF-IVP) as a protein source in culture. Cortisol was associated positively with weight in AI and negatively with body length in males. Thyroxine decreased with age, and it was positively associated with thoracic circumference in RF-IVP. Insulin-like growth factor-1 was greater in RF-IVP than in C-IVP, and it was positively associated with body length and withers height. Growth hormone was greater in females than in males and RF-IVP compared to AI and C-IVP. In conclusion, we present here the first datasets on growth parameters and growth-influencing hormones in cattle from birth to 4 years of age without observing major evidence of differences depending on the embryo origin.
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Fertilization outcome could be regulated by binding of oviductal plasminogen to oocytes and by releasing of plasminogen activators during interplay between gametes
(2012-02-02) Grullón, LA; Mondéjar Corbalán, Irene; García Vázquez, Francisco Alberto; Romar Andrés, Raquel; Coy Fuster, Pilar; Coy Fuster, Pilar; Fisiología; Facultades de la UMU
Objective: To detect plasminogen and plasminogen activators (PA) in oviduct and oocytes and to clarify the role of the plasminogen/plasmin system on mammalian fertilization. Design: Experimental prospective study. Setting: Mammalian reproduction research laboratory. Animal(s): Oviducts and ovaries from porcine and bovine females were collected at slaughterhouse. A total of 52 oviducts and 2,292 oocytes were used. Boar and bull ejaculated spermatozoa were also used. Intervention(s): Plasminogen concentration in oviductal fluid (OF) through the cycle was measured. Immunolocalization of plasminogen and PAs in oocytes was carried out before and after fertilization. Porcine and bovine oocytes were in vitro fertilized, with plasminogen and plasmin added to the culture medium at different concentrations. Main Outcome Measure(s): Plasminogen concentration in OF. Plasminogen and PAs immunolocalization in oocytes. Penetration and monospermy rates, number of spermatozoa in the ooplasma and on the zona pellucida (ZP) after IVF. Result(s): Oviductal fluid contains about 92 mg/mL of plasminogen. The mature oocyte shows immunoreactivity toward plasminogen and toward PAs on its oolemma and ZP. After fertilization, plasminogen and PAs immunolabeling decreases in the oocyte, suggesting its conversion into plasmin. When exogenous plasminogen is added to the IVF medium, sperm entry into the oocyte is hampered, suggesting that the role of plasminogen activation during fertilization is to reduce the number of (or to select) penetrating spermatozoa. Conclusion(s): The plasminogen/plasmin system is activated during gamete interaction and regulates the sperm entry into the oocyte
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The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by oviductal fluid
(2014-01-04) Mondéjar Corbalán, Irene; Avilés Sánchez, Manuel; Coy Fuster, Pilar; Coy Fuster, Pilar; Fisiología
study question: Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? summary answer: ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. what is known already: Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). study design, size, duration: Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. materials, setting, methods: In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. main results and the role of chance: rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P , 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. limitations, reasons for caution: Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. wider implications of the findings: Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed.
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Oviduct-specific glycoprotein and heparin modulate sperm-zona pellucida interaction during fertilization and contribute to the control of polyspermy
(2008-10-14) Saavedra, MD; Grullón, LA; Coy Fuster, Pilar; Cánovas Bernabé, Sebastián; Mondéjar Corbalán, Irene; Romar Andrés, Raquel; Matas Parra, Carmen; Avilés Sánchez, Manuel; Fisiología
Polyspermy is an important anomaly of fertilization in placental mammals, causing premature death of the embryo. It is especially frequent under in vitro conditions, complicating the successful generation of viable embryos. A block to polyspermy develops as a result of changes after sperm entry (i.e., cortical granule exocytosis). However, additional factors may play an important role in regulating polyspermy by acting on gametes before sperm-oocyte interaction. Most studies have used rodents as models, but ungulates may differ in mechanisms preventing polyspermy. We hypothesize that zona pellucida (ZP) changes during transit of the oocyte along the oviductal ampulla modulate the interaction with spermatozoa, contributing to the regulation of polyspermy. We report here that periovulatory oviductal fluid (OF) from sows and heifers increases (both, con- and heterospecifically) ZP resistance to digestion with pronase (a parameter commonly used to measure the block to polyspermy), changing from digestion times of approximately 1 min (pig) or 2 min (cattle) to 45 min (pig) or several hours (cattle). Exposure of oocytes to OF increases monospermy after in vitro fertilization in both species, and in pigs, sperm-ZP binding decreases. The resistance of OF-exposed oocytes to pronase was abolished by exposure to heparin-depleted medium; in a medium with heparin it was not altered. Proteomic analysis of the content released in the heparin-depleted medium after removal of OF-exposed oocytes allowed the isolation and identification of oviduct-specific glycoprotein. Thus, an oviduct-specific glycoprotein-heparin protein complex seems to be responsible for ZP changes in the oviduct before fertilization, affecting sperm binding and contributing to the regulation of polyspermy.
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The Oviduct: Functional Genomic and Proteomic Approach
(2012-06-11) Acuña, OS; Mondéjar Corbalán, Irene; Izquierdo Rico, María José; Coy Fuster, Pilar; Avilés Sánchez, Manuel; Avilés Sánchez, Manuel; Biología Celular e Histología; Facultades de la UMU
The mammalian oviduct is an anatomical part of the female reproductive tract, which plays several important roles in the events related to fertilization and embryo development. This review examines and compares several studies related to the proteomic and transcriptomic profile of the oviduct in different domestic animals. This information could be important for clarifying the role of oviductal factors in different events regulating fertilization and early embryo development, as well as for improving synthetic media for in vitro maturation ⁄ in vitro fertilization ⁄ embryo culture techniques (IVM ⁄ IVF ⁄ EC).
Open Access
Mimicking the temperature gradient between the sow’s oviduct and uterus improves in vitro embryo culture output
(Oxford University Press, 2020-07-09) García Martínez, Soledad; Latorre Reviriego, Rafael Manuel; Sánchez-Hurtado, M. A.; Sánchez-Margallo, F. M.; Bernabò, N.; Romar Andrés, Raquel; López Albors, Octavio Miguel; Coy Fuster, Pilar; Anatomía y Anatomía Patológica Comparada
This work was designed to determine temperature conditions within the reproductive tract of the female pig and study their impact on ARTs. Temperatures were recorded using a laparo-endoscopic single-site surgery assisted approach and a miniaturized probe. Sows and gilts were used to address natural cycle and ovarian stimulation treatments, respectively. According to in vivo values, IVF was performed at three temperature conditions (37.0°C, 38.5°C and 39.5°C) and presumptive zygotes were cultured in these conditions for 20 h, while further embryo culture (EC) (21–168 h post-insemination) was maintained at 38.5°C. After 20 h, different fertility parameters were assessed. During EC, cleavage and blastocyst stages were evaluated. Sperm membrane fluidity at the experimental temperatures was studied by using differential scanning calorimetry and fluorescence recovery after photobleaching techniques. An increasing temperature gradient of 1.5°C was found between the oviduct and uterus of sows (P < 0.05) and when this gradient was transferred to pig in vitro culture, the number of poly-nuclear zygotes after IVF was reduced and the percentage of blastocysts was increased. Moreover, the temperature transition phase for the boar sperm membrane (37.0°C) coincided with the temperature registered in the sow oviduct, and sperm membranes were more fluid at 37.0°C compared with those of sperm incubated at higher temperatures (38.5°C and 39.5°C). These data suggest that there may be an impact of physiological temperature gradients on human embryo development.
Open Access
Differences in weight, hierarchy, and incidence of lameness between two groups of adult pigs derived from assisted reproductive technologies
(MDPI, 2022-12-17) Ramírez, Lisette L.; Ortin Bustillo, Alba; Ramis, Guillermo; Romar, Raquel; Coy, Pilar; Romero Aguirregomezcorta, Jon; Ramírez, Lisette L.; Ortín Bustillo, Alba; Ramis Vidal, Manuel Guillermo; Romar Andrés, Raquel; Coy Fuster, Pilar; Anatomía y Anatomía Patológica Comparadas ; Facultad de Veterinaria
The in vitro production (IVP) and subsequent transfer of embryos (ET) to recipient mothers is not yet an established reproductive technology in the pig industry, as it is in cattle. However, that the trade of IVP-cryopreserved pig embryos is expected to start in the next decades. Society and governments are increasingly aware of the repercussions that IVP could have for animal health, welfare, behavior, or food safety, but proven scientific information for this type of animal does not exist, since no colonies of pigs have been created to this end. We created a small one and studied the differences between 16 IVP-derived pigs and 14 pigs derived from artificial insemination (AI), at 3.5 years of age, conceived from the same boar, and housed and fed under the same conditions since they were born. Incidence of lameness, position in the herd hierarchy, weight, adenosine deaminase activity, and hematological and biochemical analytes were compared between the two groups of animals. The results showed that the IVP animals weighed more, occupied higher positions in the herd hierarchy, and had a lower incidence of lameness. Although genetic differences from the maternal line could explain some of these results, it is also possible that the IVP animals developed better adaptative abilities, but more studies with a higher number of animals are necessary to reach consistent conclusions.