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Animals (Basel)
2020 Feb 11;102:. doi: 10.3390/ani10020276.
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Ashwin Gene Expression Profiles in Oocytes, Preimplantation Embryos, and Fetal and Adult Bovine Tissues.
Moreno-Brito V
,
Morales-Adame D
,
Soto-Orduño E
,
González-Chávez SA
,
Pacheco-Tena C
,
Espino-Solis GP
,
Leal-Berumen I
,
González-Rodríguez E
.
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The ashwin gene, originally identified in Xenopuslaevis, was found to be expressed first in the neural plate and later in the embryonic brain, eyes, and spinal cord. Functional studies of ashwin suggest that it participates in cell survival and anteroposterior patterning. Furthermore, ashwin is expressed zygotically in this species, which suggests that it participates in embryonic development. Nevertheless, the expression of this gene has not been studied in mammals. Thus, the aim of this study was to analyze the ashwin expression pattern in bovine fetal and adult tissues, as well as in three independent samples of immature and mature oocytes, and in two- to four-, and eight-cell embryos, morula, and blastocysts. Spatiotemporal expression was analyzed using real-time polymerase chain reaction (PCR); ashwin mRNA was detected in all tissues analyzed, immature and mature oocytes, and two- to eight-cell embryos. It was down-regulated in morula and blastocysts, suggesting that this expression profile is similar to that of maternal genes. Immunohistochemical localization of the ashwin protein in fetal and adult ovaries and testes reveals that this protein is consistently present during all stages of follicular development and during bovine spermatogenesis. These observations lead us to propose ashwin as an important gene involved in mammalian reproduction.
Figure 1. Ashwin expression profiles in fetal and adult tissues. (a,b) Agarose gel electrophoresis polymerase chain reaction (PCR) targeting genes encoding for ashwin and β-actin at the fetal and adult stages, respectively; (c,d) quantitative PCR results for ashwin gene expression in different tissues at the fetal and adult stages, respectively; (e) relative expression at the fetal and adult stages. Data were analyzed using a two-sample Student’s t-test. Data are presented as means ± standard deviations from two independent quantitative real-time PCR experiments (average of three independent reverse transcription reactions, each tested with three PCRs). Dunnett multiple comparison tests were performed, and expression in fetal and adult brain tissues served as a reference. Ashwin expression levels were normalized using the 2-ΔΔCT method.
Figure 2. Expression of the ashwin gene in bovine embryos at different stages, normalized to expression levels of the ashwin gene using the 2-ΔΔCT method. Expression in immature oocytes served as a reference. Dunnett multiple comparison tests were performed.
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