Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice

Abstract

Background Studies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown. Methods Female C57BL/6 mice (6–8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription–quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions. Results Embryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor. Conclusions Sperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

THC and sperm: Impact on fertilization capability, pre-implantation in vitro development and epigenetic modifications

“…Interestingly, miR-34b/c levels in sperm were only associated with a decrease in miscarriages, and increased implantation and pregnancy rates at certain Ct thresholds, determined using receiver operating characteristic curve (ROC) analysis [ 171 ]. Mechanistically, miR-34c-related improvements to pre-implantation embryo development appear to be stage-dependent and occur through diverse biological processes [ 172 ].…”

“…Considering that AR expression is critical for Sertoli cell maturation, blood-testis-barrier (BTB) formation and maintenance, germ cell proliferation and differentiation, THC-induced reduction in miR-346 transcripts may also explain the association between cannabis use and lower sperm counts [ 177 ]. Considering that the contribution and abundance of sperm-borne miRs during pre-implantation embryo development are stage-dependent [ 172 ], it is possible that we found no significant differences in the abundance of miRs-324, and -34c in blastocysts generated from THC-treated sperm because we quantified miRs only at the blastocyst stage. Although we analyzed RNA from whole blastocysts, differentially expressed genes predictive of pregnancy success appear to be more abundant within the TE compared to the ICM in human blastocysts [ 178 ].…”

Specific microRNAs in stallion spermatozoa are potential biomarkers of high functionality

“…Studies of human and other mammalian spermatozoa have found that concentrations of specific mRNAs and miRNAs are correlated with fertility and the growth of the offspring (Hamatani, 2012;Hamilton et al, 2022;Joshi et al, 2023;Zhang et al, 2019). For example, spermatozoa-borne or microinjected miR-34c enhances the development of early embryos in humans, cows, rabbits, and mice (Cui et al, 2023). We hypothesize that more dense stallion spermatozoa carry a population of miRNAs that reflects their superior ability to produce healthy embryos compared to less dense spermatozoa.…”

“…However, spermatozoa from bulls with higher fertility have lower concentrations of miR-34c than spermatozoa from bulls with lower fertility (Fagerlind et al, 2015;Kasimanickam et al, 2022). In human, cow, rabbit, and mouse species, sperm-borne or microinjected miR-34c enhances embryo development (Cui et al, 2023). A mouse study determined that a double knockout of both miR-34c and miR-449a…”

MicroRNAs in spermatogenesis dysfunction and male infertility: clinical phenotypes, mechanisms and potential diagnostic biomarkers

“…The expression of miR-34c in sperm correlates with embryonic development kinetics and clinical outcomes. Therefore, miR-34c holds promise as a potential indicator of IVF success and can provide valuable insights into embryonic development ( 140 ). A previous study demonstrated that the expression of miR-34c,-140,-21, and -375 in fresh sperm was lower in patients undergoing IVF with an average embryo score <8 than in those with an embryo score >8.…”