MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle(†)

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo–maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.