Capillary electroporation affects the expression of miRNA-122-5p from bull sperm cells

Silva, Lucas dos Santos da; Domingues, William Borges; Barreto, Bruna; Martins, Amanda Weege da Silveira; Dellagostin, Eduardo N.; Komninou, Eliza Rossi; Corcini, Carine Dahl; Varela, Antônio Sérgio; Campos, Vinícius Farias

doi:10.1016/j.gene.2020.145286

Cited by 9 publications

(4 citation statements)

References 35 publications

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“…Thus, ROS can play a role in regulating proteins involved in exocytosis [31]. The current study also demonstrated that incubation of sperm in the presence of plasmid had no effect on sperm functional parameters, which agrees with the investigations of many groups of researchers [45][46][47][48][49][50][51].…”

Section: Treatmentssupporting

confidence: 91%

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Moradbeigi,

Hosseini,

Salehi

et al. 2024

Biol Proced Online

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Background Generating targeted mutant mice is a crucial technology in biomedical research. This study focuses on optimizing the CRISPR/Cas9 system uptake into sperm cells using the methyl β-cyclodextrin-sperm-mediated gene transfer (MBCD-SMGT) technique to generate targeted mutant blastocysts and mice efficiently. Additionally, the present study elucidates the roles of cholesterol and reactive oxygen species (ROS) in the exogenous DNA uptake by sperm. Results In this study, B6D2F1 mouse sperm were incubated in the c-TYH medium with different concentrations of MBCD (0, 0.75, 1, and 2 mM) in the presence of 20 ng/µl pCAG-eCas9-GFP-U6-gRNA (pgRNA-Cas9) for 30 min. Functional parameters, extracellular ROS, and the copy numbers of internalized plasmid per sperm cell were evaluated. Subsequently, in vitro fertilization (IVF) was performed and fertilization rate, early embryonic development, and transfection rate were assessed. Finally, our study investigated the potential of the MBCD-SMGT technique in combination with the CRISPR-Cas9 system, referred to as MBCD-SMGE (MBCD-sperm-mediated gene editing), for generating targeted mutant blastocysts and mice. Results indicated that cholesterol removal from the sperm membrane using MBCD resulted in a premature acrosomal reaction, an increase in extracellular ROS levels, and a dose-dependent influence on the copy numbers of the internalized plasmids per sperm cell. Moreover, the MBCD-SMGT technique led to a larger population of transfected motile sperm and a higher production rate of GFP-positive blastocysts. Additionally, the current study validated the targeted indel in blastocyst and mouse derived from MBCD-SMGE technique. Conclusion Overall, this study highlights the significant potential of the MBCD-SMGE technique for generating targeted mutant mice. It holds enormous promise for modeling human diseases and improving desirable traits in animals. Graphical Abstract

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Section: Treatmentssupporting

confidence: 91%

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Moradbeigi,

Hosseini,

Salehi

et al. 2024

Biol Proced Online

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“…MiRNAs are small, single-stranded, non-coding RNA molecules of 22–24 nucleotides that regulate gene expression. In the last decade, miRNAs have been identified in the sperm of several mammals, including humans [ 4 ], mice [ 5 ] and cattle [ 6 , 7 ]. Thus far, up to 1,085 miRNAs have been described in bovine species ( Bos taurus ) (Sanger miRBase v.22.1; www.mirbase.org , last accession: November 2022).…”

Section: Introductionmentioning

confidence: 99%

Expression of miR-138 in cryopreserved bovine sperm is related to their fertility potential

Salas-Huetos,

Ribas-Maynou,

Mateo-Otero

et al. 2023

J Animal Sci Biotechnol

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Background MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 22–24 nucleotides that regulate gene expression. In the last decade, miRNAs have been described in sperm of several mammals, including cattle. It is known that miRNAs can act as key gene regulators of early embryogenesis in mice and humans; however, little is known about the content, expression, and function of sperm-borne miRNAs in early bovine embryo. In this study, total sperm RNA was isolated from 29 cryopreserved sperm samples (each coming from a separate bull) using a RNeasy kit and treatment with DNase I. RNA concentration and purity were determined through an Epoch spectrophotometer and an Agilent Bioanalyzer. The expression of 10 candidate miRNAs in bovine sperm (bta-miR-10a, bta-miR-10b, bta-miR-138, bta-miR-146b, bta-miR-19b, bta-miR-26a, bta-miR-34a, bta-miR-449a, bta-miR-495 and bta-miR-7), previously identified in testis and/or epididymis, was evaluated with RT-qPCR. The cel-miR-39-3p was used as a spike-in exogenous control. Nonparametric Mann–Whitney tests were run to evaluate which miRNAs were differentially expressed between bulls with high fertility [HF; non-return rates (NRR) ranging from 39.5 to 43.5] and those with subfertility (SF; NRR ranging from 33.3 to 39.3). Several sperm functionality parameters (e.g., viability, membrane stability or oxygen consumption, among others) were measured by multiplexing flow cytometry and oxygen sensing technologies. Results RNA concentration and purity (260/280 nm ratio) (mean ± SD) from the 29 samples were 99.3 ± 84.6 ng/µL and 1.97 ± 0.72, respectively. Bioanalyzer results confirmed the lack of RNA from somatic cells. In terms of the presence or absence of miRNAs, and after applying the Livak method, 8 out of 10 miRNAs (bta-miR-10b, -138, -146b, -19b, -26a, -449a, -495, -7) were consistently detected in bovine sperm, whereas the other two (bta-miR-10a, and -34a) were absent. Interestingly, the relative expression of one miRNA (bta-miR-138) in sperm was significantly lower in the SF than in the HF group (P = 0.038). In addition to being associated to fertility potential, the presence of this miRNA was found to be negatively correlated with sperm oxygen consumption. The expression of three other miRNAs (bta-miR-19b, bta-miR-26a and bta-miR-7) was also correlated with sperm function variables. Conclusions In conclusion, although functional validation studies are required to confirm these results, this study suggests that sperm bta-miR-138 is involved in fertilization events and beyond, and supports its use as a fertility biomarker in cattle.

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“…Further, among the most abundant miRNAs, the let-7 family and miRNA-146 were found to regulate the differentiation of spermatogonia and impaired blastocyst implantation in mice whereas, miRNA-16 is known to coordinate cell-cycle ( Zhang et al, 2012 ). A recent study suggests miRNA (e.g., miR-126-5p) accumulates during the final stage of spermatogenesis, and epidydimal transit and their expression is affected during heat stress, which might explain reduced fertility in heat-stressed bulls due to impaired spermatogenesis and maturation ( Dos Santos da Silva et al, 2021 ). The miR-17-92 and miR-106b-25 are two miRNA clusters whose target genes were reported to be essential for the progression of spermatogenesis ( Tong et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock

et al. 2021

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Livestock production contributes to a significant part of the economy in developing countries. Although artificial insemination techniques brought substantial improvements in reproductive efficiency, male infertility remains a leading challenge in livestock. Current strategies for the diagnosis of male infertility largely depend on the evaluation of semen parameters and fail to diagnose idiopathic infertility in most cases. Recent evidences show that spermatozoa contains a suit of RNA population whose profile differs between fertile and infertile males. Studies have also demonstrated the crucial roles of spermatozoal RNA (spRNA) in spermatogenesis, fertilization, and early embryonic development. Thus, the spRNA profile may serve as unique molecular signatures of fertile sperm and may play pivotal roles in the diagnosis and treatment of male fertility. This manuscript provides an update on various spRNA populations, including protein-coding and non-coding RNAs, in livestock species and their potential role in semen quality, particularly sperm motility, freezability, and fertility. The contribution of seminal plasma to the spRNA population is also discussed. Furthermore, we discussed the significance of rare non-coding RNAs (ncRNAs) such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs) in spermatogenic events.

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Capillary electroporation affects the expression of miRNA-122-5p from bull sperm cells

Cited by 9 publications

References 35 publications

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Methyl β-Cyclodextrin-sperm-mediated gene editing (MBCD-SMGE): a simple and efficient method for targeted mutant mouse production

Expression of miR-138 in cryopreserved bovine sperm is related to their fertility potential

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock

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