Browsing by Author "Peralta, Oscar A."

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    Bovine adipose tissue-derived mesenchymal stem cells self-assemble with testicular cells and integrates and modifies the structure of a testicular organoids
    (2024) Cortez, Jahaira; Torres, Cristian G.; Parraguez, Victor H.; De los Reyes, Monica; Peralta, Oscar A.
    Mesenchymal stem cells (MSC) display self-renewal and mesodermal differentiation potentials. These characteristics make them potentially useful for in vitro derivation of gametes, which may constitute experimental therapies for human and animal reproduction. Organoids provide a spatial support and may simulate a cellular niche for in vitro studies. In this study, we aimed at evaluating the potential integration of fetal bovine MSCs derived from adipose tissue (AT-MSCs) in testicular organoids (TOs), their spatial distribution with testicular cells during TO formation and their potential for germ cell differentiation. TOs were developed using Leydig, Sertoli, and peritubular myoid cells that were previously isolated from bovine testes (n = 6). Thereafter, TOs were characterized using immunofluorescence and Q-PCR to detect testicular cell-specific markers. AT-MSCs were labeled with PKH26 and then cultured with testicular cells at a concentration of 1 x 106 cells per well in Ultra Low Attachment U-shape bottom (ULA) plates. TOs formed by testicular cells and AT-MSCs (TOs + AT-MSCs) maintained a rounded structure throughout the 28-day culture period and did not show significant differences in their diameters. Conversely, control TOs exhibited a compact structure until day 7 of culture, while on day 28 they displayed cellular extensions around their structure. Control TOs had greater (P < 0.05) diameters compared to TOs + AT-MSCs. AT-MSCs induced an increase in proportion of Leydig and peritubular myoid cells in TOs + AT-MSCs; however, did not induce changes in the overall gene expression of testicular cell-specific markers. STAR immunolabelling detected Leydig cells that migrated from the central area to the periphery and formed brunches in control TOs. However, in TOs + AT-MSCs, Leydig cells formed a compact peripheral layer. Sertoli cells immunodetected using WT1 marker were observed within the central area forming clusters of cells in TOs + AT-MSCs. The expression of COL1A associated to peritubular myoids cells was restricted to the central region in TOs + AT-MSCs. Thus, during a 28-day culture period, fetal bovine AT-MSCs integrated and modified the structure of the TOs, by restricting formation of branches, limiting the overall increase in diameters and increasing the proportions of Leydig and peritubular myoid cells. AT-MSCs also induced a reorganization of testicular cells, changing their distribution and particularly the location of Leydig cells.
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    Bovine Peripheral Blood-Derived Mesenchymal Stem Cells (PB-MSCs) and Spermatogonial Stem Cells (SSCs) Display Contrasting Expression Patterns of Pluripotency and Germ Cell Markers under the Effect of Sertoli Cell Conditioned Medium
    (2024) Segunda, Moises N.; Diaz, Carlos; Torres, Cristian G.; Parraguez, Victor H.; De los Reyes, Monica; Peralta, Oscar A.
    In vitro gamete derivation has been proposed as an interesting strategy for treatment of infertility, improvement of genetic traits, and conservation of endangered animals. Spermatogonial stem cells (SSCs) are primary candidates for in vitro gamete derivation; however, recently, mesenchymal stem cells (MSCs) have also been proposed as candidates for germ cell (GCs) differentiation mainly due to their transdifferentiating capacity. The objective of the present study was to compare the potential for GC differentiation of bovine peripheral blood-derived MSCs (PB-MSCs) and SSCs under the effect of conditioned medium (CM) derived from Sertoli cells (SCs/CM). Samples were collected every 7 days for 21 days and analyzed for pluripotent, GC, and MSC marker expression. The absence of OCT4 and the increased (p < 0.05) expression of NANOG seems to play a role in SSC differentiation, whereas the absence of NANOG and the increased expression (p < 0.05) of OCT4 may be required for PB-MSC differentiation into GCs. SSCs cultured with SCs/CM increased (p < 0.05) the expression of PIWIL2 and DAZL, while PB-MSCs cultured under the same condition only increased (p < 0.05) the expression of DAZL. Overall, the patterns of markers expression suggest that PB-MSCs and SSCs activate different signaling pathways after exposure to SCs/CM and during differentiation into GCs.
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    Colostrum traits and newborn body weight and growth: comparison between single and twin underfed sheep pregnancies
    (2023) Turin, Jesus; Sales, Francisco; Peralta, Oscar A.; De los Reyes, Monica; Borie, Consuelo; Carrasco, Albert; Gonzalez-Bulnes, Antonio; Parraguez, Victor H.
    Maternal nutrition during gestation plays an important role in colostrum production, postnatal growth, and survival of newborn lambs, especially in twin gestations. This research aimed to investigate the effects of chronic natural undernutrition on colostrum traits and early lamb's postnatal growth born from single and twin sheep pregnancies developed in a restrictive prairie, representative of southern Patagonia. Single- and twin-bearing ewes (n = 20 per group) were maintained grazing in a natural pasture. At 140 days of gestation, ewes were placed in individual pens for lambing control. Colostrum was collected immediately after delivery and at 12, 24, and 36 h postpartum, for determination of yield and composition. Maternal blood was obtained at 140 days of gestation and at lambing for plasma glucose, progesterone, 17 beta-estradiol, and IgG determination. Newborn lamb blood for determining glycaemia and IgG was collected at birth and at 12, 24, 36, and 120 h after birth. Lamb mortality and growth was assessed from birth until 30 days of life. No differences were observed in progesterone and 17 beta-estradiol. There were no differences in colostrum yields and fat components, however single- had higher values of protein and lactose than twin-bearing ewes (p < 0.05 for both). Singletons had higher glycaemia than twins at 12 h postpartum (102.2 +/- 32.8 vs. 73.4 +/- 29.9 mg/dL, p < 0.05). Colostrum IgG content was similar at delivery but higher in single ewes at 12 and 24 h, reaching a similar values at 36 h (4.7 +/- 9.7 and 5.8 +/- 7.7 mg/mL in single and twin pregnancies, respectively). Newborn IgG was higher in singletons compared to twins at least until 48 h of life. Lams body weight was always superior in singleton than twins from birth until 30 days of life. Mortality did not differ during the first week of life, but it increased significantly only in twins until day 30 of life. Undernourishment in pregnant ewes affected colostrum quantity and quality, resulting in a lower postnatal growth and a higher mortality in twins. Alternative managements favoring fetal growth, birth weight and neonatal viability in twin sheep pregnancies are needed, when flocks are breed under harsh environments.
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    Dynamic Expression of Follicle-Stimulating Hormone and Estrogen mRNA Receptors Associated with microRNAs 34a and -let-7c in Canine Follicles during the Estrous Cycle
    (2024) De los Reyes, Monica; Dettleff, Phillip; Palomino, Jaime; Peralta, Oscar A.; Vergara, Ana
    The genes encoding for estrogen receptor (ESR2) and follicle-stimulating hormone receptor (FSHR) play crucial roles in ovarian follicular development. This study aimed to determine the expression levels of miRNAs predicted against FSHR and ESR2 mRNAs in follicular cells related to their target genes during the estrous cycle in canines. Antral follicles were dissected from 72 ovaries following ovariohysterectomies. MiRNAs regulating FSHR and ESR2 genes were selected from miRNA databases, and mature miRNA and mRNA expression profiling was performed using real-time polymerase chain reaction (PCR). The best miRNA for each target gene was selected considering the quantitative PCR (qPCR) performance and target prediction probability, selecting only miRNAs with a binding p-value of 1.0, and choosing cfa-miR-34a and cfa-let-7c for FSHR and ESR2, respectively. The expression levels comparing the different phases of the estrous cycle were evaluated using ANOVA. Pearson correlations between the expression pattern of each miRNA and their target genes were performed. Each miRNA and its target genes were expressed in the granulosa cells in all estrous phases. FSHR remained low in anestrus and proestrus, increased (p < 0.05) to the highest level in estrus, and decreased (p < 0.05) in diestrus. ESR2 showed the same trend as FSHR, with the highest (p < 0.05) expression in estrus and the lowest (p < 0.05) in anestrus and proestrus. A tendency for an inverse relationship was observed between the expression of miR-34a and FSHR only in the anestrus phase, while an inverse correlation (r = -0.8) was found between miRNA-7c and ESR2 (p < 0.01). The expression profile of miR-34a and miR-let-7c and their predicted target genes of dog ovarian follicles throughout the estrous cycle observed in this study suggest a role in the transcriptional regulation of FSHR and ESR2, which is the first evidence of the involvement of these miRNAs in the canine follicular function.
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    Early apoptosis detection in canine granulosa cells through the analysis of BCL-2 and BAX proteins during the follicular development associated with oocyte maturation
    (2024) Salinger, Michelle; Palomino, Jaime; Peralta, Oscar A.; Parraguez, Victor H.; de los Reyes, Monica
    The objective of this study was to investigate the early follicular apoptosis in canine ovarian follicles by examining the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX proteins throughout the estrous cycle associated with oocyte maturation. Follicular cells from preantral and antral follicles of varying sizes were isolated and grouped based on follicle type and estrous phase. Antral follicles underwent flow cytometry analysis, whereas preantral follicles were subjected to Western blotting. The meiotic capacity of oocytes from these different follicle types was evaluated through in vitro maturation. Statistical analysis included ANOVA and Duncan's test. Results showed fluctuations in BCL-2 and BAX levels across different follicular stages and estrous phases. BCL-2 levels increased (P<0.05) with follicular development in antral follicles, particularly during estrus, while BAX exhibited variations peaking (P<0.05) during estrus. The BCL-2/ BAX ratio in antral follicles was higher (P<0.05) in estrus and diestrus compared to anestrus and proestrus. Additionally, BCL-2 and BAX proteins were detected in preantral follicles, with varying expression levels (P<0.05) across estrous phases. The BCL-2/BAX ratio in preantral follicles was highest (P<0.05) during anestrus and estrus and decreased (P<0.05) in proestrus and diestrus. Oocytes from preantral follicles did not reach the MII stage, regardless of the levels of BCL-2/BAX. Conversely, oocytes obtained from large follicles during estrus showed the highest (P<0.05) maturation percentages, which were associated with the highest BCL-2/BAX ratio. These findings provide insights into the dynamic patterns of BCL-2 /BAX in canine follicles across the estrous cycle, shedding light on their potential roles in oocyte development.
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    Effect of Melatonin on Chemoresistance Exhibited by Spheres Derived from Canine Mammary Carcinoma Cells
    (2024) Cataldo, Dania; Aravena, Guillermo; Escobar, Alejandro; Tapia, Julio C.; Peralta, Oscar A.; Torres, Cristian G.
    Mammary cancer is a frequent disease in female dogs, where a high proportion of cases correspond to malignant tumors that may exhibit drug resistance. Within the mammary tumor microenvironment, there is a cell subpopulation called cancer stem cells (CSCs), which are capable of forming spheres in vitro and resisting anti-tumor treatments, partly explaining the recurrence of some tumors. Previously, it has been described that spheres derived from canine mammary carcinoma cells CF41.Mg and REM 134 exhibit stemness characteristics. Melatonin has shown anti-tumor effects on mammary tumor cells; however, its effects have been poorly evaluated in canine mammary CSCs. This study aimed to analyze the effect of melatonin on the chemoresistance exhibited by stem-like neoplastic cells derived from canine mammary carcinoma to cytotoxic drugs such as doxorubicin and mitoxantrone. CF41.Mg and REM 134 cells were cultured in high-glucose DMEM supplemented with fetal bovine serum and L-glutamine. The spheres were cultured in ultra-low attachment plates in DMEM/F12 medium without fetal bovine serum and with different growth factors. The CD44(+)/CD24(-/low) phenotype was analyzed by flow cytometry. The viability of sphere-derived cells (MTS reduction) was studied in the presence of melatonin (0.1 or 1 mM), doxorubicin, mitoxantrone, and luzindole. In addition, the gene (RT-qPCR) of the multidrug resistance bombs MDR1 and ABCG2 were analyzed in the presence of melatonin. Both cell types expressed the MT1 gene, which encodes the melatonin receptor MT1. Melatonin 1 mM does not modify the CD44(+)/CD24(-/low) phenotype; however, the hormone reduced viability (p < 0.0001) only in CF41.Mg spheres, without inducing an additive effect when co-incubated with cytotoxic drugs. These effects were independent of the binding of the hormone to its receptor MT1, since, by pharmacologically inhibiting them, the effect of melatonin was not blocked. In CF41.Mg spheres, the relative gene expression of ABCG2 and MDR1 was decreased in response to the hormone (p < 0.001). These results indicate that melatonin negatively modulates the cell survival of spheres derived from CF41.Mg cells, in a way that is independent of its MT1 receptor. These effects did not counteract the resistance to doxorubicin and mitoxantrone, even though the hormone negatively regulates the gene expression of MDR1 and ABCG2.
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    Potential of mesenchymal stromal/stem cells and spermatogonial stem cells for survival and colonization in bull recipient testes after allogenic transplantation
    (2024) Segunda, Moises N.; Cortez, Jahaira; Diaz, Carlos; Arancibia, Richard; Torres, Cristian G.; Parraguez, Victor H.; de los Reyes, Monica; Peralta, Oscar A.
    Stem cell transplantation into seminiferous tubules of recipient testis could become a tool for fertility restoration, genetic improvement, or conservation of endangered species. Spermatogonial stem cells (SSCs) are primary candidates for transplantation; however, limited abundance, complexity for isolation and culture, and lack of specific markers have limited their use. Mesenchymal stromal/stem cells (MSCs) are multipotent progenitors that are simple to isolate and culture and possess specific markers for identification, and immune evasive and migratory capacities. The objective of the present study was to evaluate the potential for survival and colonization in seminiferous tubules of two different concentrations of bovine fetal adipose tissue-derived MSCs (ATMSCs), native of pre-induced, and to compare the fate of bovine adult peripheral blood-derived MSCs (PB-MSCs) and SSCs after allogenic transplantation in testis of recipient bulls. In experiment 1, AT-MSCs at two concentrations (1x107 and 2x107; n = 3) or pre-exposed to 2 mu M testosterone and 1 mu M retinoic acid (RA) for 14 days (n = 5) were evaluated. In experiment 2, adult PB-MSCs and SSCs (4x107 cells each) pre-exposed to Sertoli cell conditioned media (SCs/CM; n = 4) for 14 days were compared. Each cell type was separately labelled with PKH26 and then transplanted into testes of 8-month-old recipient bulls. Four weeks (Exp. 1) and two weeks (Exp. 2) after transplantation, testicular tissue was processed for confocal microscopy detection of PKH26-positive cells. Mean number of PKH26-positive cells were higher (P < 0.05) in testis transplanted with 2x107 AT-MSCs in the proximal (6.7 f 3.7) and medial (6.6 f 3.2) sections compared to testis transplanted with 1x107 ATMSCs (proximal: 1.9 f 1; medial: 1.9 f 1) sections or pre-induced AT-MSCs (proximal: 4.7 f 5.6; medial: 3.8 f 4.1). In Exp. 2, mean number of PKH26-positive SSCs in medial testicular section (22.5 f 1.3) were higher (P <0.05) compared to respective section in PB-MSCs group (17 f 4.2). Thus, in vivo data indicates that a higher number of transplanted AT-MSCs resulted in more cells surviving and colonizing seminiferous tubules; however, pre-induction with testosterone and RA did not improve these capacities. SSCs displayed a greater capacity for survival and colonization in recipient seminiferous tubules; however, PB-MSCs were observed in all sections of testis after two weeks of transplantation.