Browsing by Author "Palomino, Jaime"

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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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    Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi
    (2021) Palomino, Jaime; Gomez, Camila; Otarola, Maria Teresa; Dettleff, Phillip; Patino-Garcia, Daniel; Orellana, Renan; Moreno, Ricardo D.
    In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.
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    Estetrol Increases Progesterone Genetic Response without Triggering Common Estrogenic Effects in Endometriotic Cell Lines and Primary Cultures
    (2023) Patino-Garcia, Daniel; Palomino, Jaime; Pomes, Cristian; Celle, Claudia; Torres-Estay, Veronica; Orellana, Renan
    Estetrol (E4), a natural estrogen produced by the human fetal liver, is actively studied for menopause and breast cancer treatment. It has low side effects and preferential estrogen receptor alpha (ER alpha) affinity. There are no data about its effects on endometriosis, a common gynecological disease affecting 6-10% of cycling women, generating painful pelvic lesions and infertility. Current combined hormone treatment (progestins and estrogens) is safe and efficient; nevertheless, one-third of patients develop progesterone (P4) resistance and recurrence by reducing P4 receptors (PRs) levels. We aimed to compare E4 and 17 beta-estradiol (E2) effects using two human endometriotic cell lines (epithelial 11Z and stromal Hs832 cells) and primary cultures from endometriotic patients. We evaluated cell growth (MTS), migration (wound assay), hormone receptors levels (Western blot), and P4 response by PCR array. Compared to E2, E4 did not affect cell growth or migration but increased estrogen receptor alpha (ER alpha) and PRs, and reduced ER beta. Finally, the incubation with E4 improved the P4 gene response. In conclusion, E4 increased PRs levels and genetic response without inducing cell growth or migration. These results suggest that E4 might be useful for endometriosis treatment avoiding P4 resistance; however, evaluating its response in more complex models is required.
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    High temperature induces oxidative damage, immune modulation, and atrophy in the gills and skeletal muscle of the teleost fish black cusk-eel (Genypterus maculatus)
    (2025) Becerra, Sofía; Arriagada-Solimano M.; Escobar Aguirre, Sebastián Gonzalo; Palomino, Jaime; Aedo, Jorge; Estrada, Juan Manuel; Barra Valdebenito, Verónica; Zuloaga, Rodrigo; Valdés, Juan Antonio; Dettleff, Phillip
    The high temperature associated with heat waves is a relevant abiotic factor that could impact the biology of teleost fish. The innate immune response, muscular growth, and oxidative stress status are relevant functions in fish tissues that could be affected by increased temperature. In this study, black cusk-eel (Genypterus maculatus) juveniles were subjected to increased temperature, to experimentally replicate heat waves registered from the South Pacific Ocean for five days. The results showed that thermal stress modulated the immune response in gills, with up-regulation of antibacterial peptides, pro-inflammatory cytokines, and Toll-like receptors genes, including hepcidin, gzma, tnfa, cxcl8, and tlr5, with no effect on complement system genes. In skeletal muscle, high temperature triggered atrophy-related gene expression, with up-regulation of foxo1, foxo3, fbxo32, murf1, and atg16l. Increased temperature also generated an up-regulation of transcripts encoding heat shock protein (hsp60 and hsp70) in gills and skeletal muscle, generating oxidative stress in both tissues, with increased expression of the antioxidant genes sod1 and gpx1 in gills and skeletal muscle, respectively, with oxidative damage observed at the DNA level (AP sites), protein (carbonyl content), and lipoperoxidation (HNE content) in both tissues. The present study shows that short-term increases in temperature like those observed in heat waves could affect the immune response in gills, induced atrophy in skeletal muscle, and generate oxidative stress in a teleost species important for Chilean aquaculture diversification, information relevant under the context of climate change scenario.
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    Reproductive alterations in chronically exposed female mice to environmentally relevant doses of a mixture of phthalates and alkylphenols
    (2018) Patiño García, Daniel; Cruz Fernandes, Leonor; Buñay, Julio; Palomino, Jaime; Moreno Mauro, Ricardo D.
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    Thermal stress generate oxidative damage in black cusk-eel embryos (Genypterus maculatus)
    (WILEY, 2021) Dettleff, Phillip; Zuloaga, Rodrigo; Godoy, Camila; Palomino, Jaime; Molina, Alfredo; Antonio Valdes, Juan