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

Abstract

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.

Elucidating the Role of OXPHOS Variants in Asthenozoospermia: Insights from Whole Genome Sequencing and an In Silico Analysis

“…Furthermore, miR-21 has been shown to regulate the self-renewal of mouse spermatogonial stem cells [44], with potential implications for spermatogenesis also reported in other animal studies [45,46]. MiR-495, recognized also for its role in reproduction through various animal studies [47][48][49], is expressed in the testis [47] and has been associated with the progression of human pregnancy [50], as well as mitochondrial metabolism [51]. Additionally, miR-329 is expressed in rat Leydig cells and plays a role during development from the progenitor to the adult stage [52].…”

Cryopreservation induces changes in the expression of noncoding RNAs in semen: a mini-review

“…In addition to the abovementioned miRNAs, miR-138 was shown to be expressed at significantly lower levels in the subfertile population (according to the nonreturn rate) than in the highly fertile population after thawing; furthermore, the expression of miR-138 was negatively correlated with sperm oxygen consumption [24]. Since cryopreserved sperm is predominantly used for cattle insemination, bovine sperm cryopreservation is important from the perspective of scientific research and has important commercial value.…”