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Sexual dimorphism of bovine blastocysts has previously been observed through differences in development, cell death, metabolism, telomere length, DNA methylation, and transcriptomics. However, dimorphism in the secretion of miRNAs to culture media has not yet been evaluated. The objectives of this study were to determine if sex-specific blastocyst miRNA secretion occurs and to further investigate the role these miRNAs may have in the interaction between a blastocyst and the maternal environment.
Mammalian embryos exhibit sexual dimorphism in development, genetics, and epigenetics (
When cultured under the same conditions, embryos exhibit sexual dimorphism in up to one-third of actively expressed genes (
To fully understand successful pregnancy, it is important to elucidate the signaling mechanisms, or dialog, between an embryo and the mother. A potential signaling mechanism involved in regulation of embryo development is the secretion of miRNAs into the extracellular environment. miRNAs are a family of short, single-stranded, non-coding RNAs that are approximately 19–25 nucleotides in length. These small molecules modulate sequence-specific mRNA transcription, leading to regulation of gene expression (
Embryo-secreted miRNAs have demonstrated potential to affect endometrial transcriptomes. For example, miR-30b transfected into human endometrial epithelial cells induced transcriptomic changes in the cells (
This study is exempt from approval of the institutional and national requirements of Animal Care and Use Committee because cows used for
Embryos and media were procured as described by
Gametes were co-cultured with sperm for 20 h at which point presumptive zygotes were stripped of their cumulus cells and washed in supplemented Vigro TL-Hepes (Bioniche). The presumptive zygotes were placed 25 per drop into a 50 μL drop of CR1aa culture media (
On day 8 of development, individually cultured embryos were morphologically assessed for characteristics of the blastocyst stage of development. Embryos which developed a blastocoel cavity and demonstrated a distinct inner cell mass and outer trophectoderm were deemed blastocysts, whereas those which failed to develop to the blastocyst stage were deemed degenerate embryos. Blastocysts that were at the mid-to-expanded blastocyst stage and quality grades 1 and 2 (
Individual embryos were genotyped for sex determination as described by
For miRNA differential expression analysis between female and male embryos, a total of three pools of media were generated each for males (each pool consisted of media from 10 embryos) and females (each pool consisted of media from 9 embryos). Embryo pools were determined following IVF completion. Each pool included media from a unique set of individual embryos. Pools were designed to contain media of embryos from all three IVF runs, representing both sires equivalently across pools. RNA was extracted from each pool using a miRNeasy Serum/Plasma kit (Qiagen, Germantown, MD, USA). A total of three extractions were carried out per pool, with an initial input volume of 120–140 μL media sample, consisting of an equal volume of eluent from individual embryos. miRNA profiling was performed using the Firefly® Circulating miRNA Assay Immunology Panel (ABCAM, Cambridge, MA, USA). The immunology panel includes 68 immune response-related miRNAs chosen by ABCAM based on known functions related to immune response and differential regulation in plasma or serum. We hypothesized that these miRNAs could play a role in the immune response of the mother to the developing embryo. Complementary oligonucleotides which encode hydrogel microparticles were hybridized to selected miRNAs. The oligonucleotide adapter served as a universal PCR priming site, allowing for fluorescent amplification of the target. Amplified products were then re-hybridized to original oligonucleotide particles, and an EMD Millipore Guava 8HT Flow Cytometer (Merck, Darmstadt, Germany) was used to quantify the hybridization. Measurements were performed in triplicates.
Statistical analysis of differentially expressed miRNAs was performed using the Firefly® Analysis Workbench Software (ABCAM) in which miRNA expression levels of male and female embryos were compared to each other. An unpaired
For the validation of differentially expressed miRNAs detected by Firefly® particle technology, three miRNAs (miR-22, miR-122, and miR-320a) were chosen for further analysis by qRT-PCR. A total of five additional IVF replicates were performed to generate 146 embryos derived from one sire and different dams. Fertilization, embryo grading, media collection, and embryo genotyping were performed as described above. A total of 101 blastocysts embryos were successfully genotyped for validation, resulting in 51 male and 50 female blastocysts (Supplementary Table
To investigate the potential of validated miRNAs as signaling molecules to maternal tissues, a primary cell culture system was implemented. Bovine endometrial epithelial cells (BEECs) (Cell Applications Inc., San Diego, CA, USA) were cultured for 6–8 passages and seeded in a 96-well culture plate at 7000 cells per cm2 per manufacturer instruction. Cells were cultured in Bovine Endometrial Epithelial Cell Growth Medium (Cell Applications Inc.) and passaged using Hank’s Balanced Salt Solution, Trypsin/EDTA and Trypsin Inhibitor (Cell Applications Inc.), as directed.
Synthetic mimics for the miRNAs miR-22, miR-122, and miR-320a (Qiagen) — specific to those found to be differentially expressed between male and female embryos — were supplemented to BEECs to assess whether these miRNAs modulate maternal gene expression. At 24 h post-passage of cells, 50 nM mimic miRNA was added to a well of a 96-well plate containing BEECs. Notably, no transfection reagent was used in order to more closely simulate an
To evaluate the effect of miRNAs on gene expression in the endometrial epithelial cells, an additional experiment was performed, in which cells were treated with 1 μL of Lipofectamine 2000 (Thermo Fisher Scientific, Madison, WI, USA) in conjunction with 50 nM of miRNA and Opti-MEM medium, as specified by the manufacturer. A control of lipofectamine-only treated cells was cultured simultaneously.
Total RNA was extracted from the BEECs using a MiRNeasy Mini Kit (Qiagen) to assess miRNA uptake by the maternal cells and expression of genes targeted by supplemented miRNAs, respectively. Reverse transcription and quantification of uptake of miRNAs in cells were performed as described for media. Reverse transcription of mRNA was carried out using an iScript cDNA kit (Bio-Rad, Hercules, CA, USA) and qRT-PCR to evaluate the expression of candidate mRNA targets was performed using iTaq Universal SYBR Green Supermix (Bio-Rad). The β-actin gene was selected as an internal control according to its stability across samples in comparison to GAPDH. Intron-spanning primers were designed for each target mRNA to avoid amplification of genomic DNA (Supplementary Table
Out of 248 blastocysts collected through eight rounds of IVF, 158 embryos were successfully genotyped, resulting in a total of 81 males and 77 females. Thus, no significant sex bias was observed in our IVF system (
To test whether embryonic miRNAs are taken up by maternal endometrial cells, synthetic miRNA mimics corresponding to miR-22, miR-122, and miR-320a were supplemented to primary endometrial epithelial cells.
To assess the impact of miRNA supplementation on gene expression changes in the BEECs, we used TargetScan
Blastocyst-secreted miRNAs may play a remarkable role in the communication of sexually dimorphic states of conceptuses to the mother. Our findings show for the first time that male and female blastocysts differentially secrete miRNAs into culture media. Furthermore, we demonstrate that these miRNAs are taken up by maternal endometrial cells. Two miRNAs targeted
No difference in the ratio of male to female blastocysts was observed in this study, which is in agreement with other reports of
Epigenetic sexual dimorphism has previously been reported through observations of differential DNA methylation (
In the present study, a total of eight miRNAs were found to be upregulated in the conditioned culture media of female embryos. Indeed, patterns observed during development indicate the secreted miRNAs upregulated in female media may serve as non-invasive biomarkers for embryo growth. The observation that miR-122 decreases from hours 0 to 22 of maturation in bovine oocytes (
Significant uptake of miR-122 and miR-320a, as well as a trend of uptake for miR-22, were observed in this study. Internalization of miRNAs by endometrial cells exhibits the ability for these molecules to interact with the maternal environment and indicates their potential to function as signaling molecules. This study showed endometrial cell uptake of miRNA at half the concentration used previously by
Mechanisms through which endometrial cells take up various miRNAs have yet to be demonstrated, though the uptake of two out of three distinct miRNAs indicates passage of miRNAs into cells may be somewhat selective. It is unknown whether endometrial cells perform uptake through various modes, given that transport of extracellular miRNAs can occur through binding of the miRNA to the AGO protein, apoptotic bodies, or extracellular vesicles (microvesicles and exosomes) (
Interestingly, the miRNAs investigated in this study have been shown to affect processes critical to the establishment of an interface between the embryo and the mother. Angiogenesis, a process necessary for embryo viability, is inhibited by miR-320 in rat myocardial microvascular endothelial cells (
Computational prediction revealed that all three miRNAs (miR-22, miR-122, and miR-320a) collectively target
Further, the substantial upregulation of the
The most well-studied aspect of
Our study was based on
Although this study was limited to a panel of miRNAs chosen specifically for their impacts on immunity as opposed to a high-throughput sequencing approach, the results provide a basis for identifying larger-scale dimorphisms of secreted miRNAs. A comprehensive understanding of the exact roles for sexually dimorphic miRNA production has not yet been attained, but future research should focus on understanding larger-scale interactions and collective targeting of subsets of miRNAs on mRNA transcripts in order to better elucidate the roles these molecules may play in maternal cells and to accommodate optimal development of blastocysts relative to their sex.
The current study demonstrates the potential for blastocysts of differing sex to produce dimorphic signals in the form of miRNAs, which can collectively impose changes in transcripts of maternal cells. Endometrial cell internalization of miRNAs, and subsequent upregulation of
NG carried out the experiments, data analysis and drafting of the manuscript. JK participated in the
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors would like to thank John Parrish and Rick Monson for guidance in the production of embryos.
The Supplementary Material for this article can be found online at: