Changes in miRNA levels of sperm and small extracellular vesicles of seminal plasma are associated with transient scrotal heat stress in bulls

Abstract

Scrotal heat stress affects spermatogenesis and impairs male fertility by increasing sperm morphological abnormalities, oxidative stress and DNA fragmentation. While sperm morpho-functional changes triggered by scrotal heat stress are well described, sperm molecular alterations remain unknown. Recently, spermatozoa were described as accumulating miRNAs during the last steps of spermatogenesis and through epididymis transit, mainly by communication with small extracellular vesicles (sEVs). Herein, the aim was to investigate the impact of scrotal heat stress in miRNAs profile of sperm, as well as, seminal plasma sEVs. Six Nelore bulls (Bos indicus) were divided into two groups: Control (CON; n = 3) and Scrotal Heat Stress (SHS; n = 3; scrotal heat stressed during 96 h by scrotal bags). The day that the scrotal bags were removed from SHS group was considered as D0 (Day zero). Seminal plasma sEVs were isolated from semen samples collected seven days after heat stress (D+7) to evaluate sEVs diameter, concentration, and 380 miRNA levels. Sperm morpho-functional features and profile of 380 miRNAs were evaluated from semen collected 21 days after heat stress (D+21). As a control, sEVs and sperm were analyzed seven days before heat stress (D-7). Only semen parameters that were not significantly different (P > 0.05) among bulls on D-7 were addressed on D+7 and D+21. While no alterations in diameter and concentration were detected in sEVs on D+7 between CON and SHS groups, three sEVs-miRNAs (miR-23b-5p, −489 and −1248) were down-regulated in SHS bulls compared to CON on D+7; other three (miR-126-5p, −656 and −1307) displayed a tendency (0.05 < P < 0.10) to be altered. Sperm oxidative stress was higher, and the level of 21 sperm miRNAs was altered (18 down-, 3 up-regulated) in SHS bulls compared to CON on D+21. Functional analysis indicated that target genes involved in transcription activation, as well as cell proliferation and differentiation were related to the 18 down-regulated sperm miRNAs (miR-9-5p, −15a, −18a, −20b, −30a-5p, −30b-5p, −30d, −30e-5p −34b, −34c, −106b, -126-5p, −146a, −191, −192, −200b, −335 and −449a). Thus, the scrotal heat stress probably impacted testicular and epididymis functions by reducing the levels of a substantial proportion of sEVs and sperm miRNAs. Our findings suggest that miR-126-5p was possibly trafficked between sEVs and sperm and provide new insights on the mechanism by which sperm acquire miRNAs in the last stages of spermatogenesis and sperm maturation in cattle.