MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.

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Sulfate is important for mammalian growth and development. During pregnancy, maternal circulating sulfate levels increase by 2-fold, enhancing sulfate availability to the fetus. We used quantitative real-time PCR to determine sulfate transporter mRNA levels during mouse gestation in three tissues: kidney and ileum, to identify transporters involved in sulfate absorption and maintaining high maternal circulating sulfate level; and placenta, to build a model of directional sulfate transport from mother to fetus. In the kidney, Slc13a1 and Slc26a1 were the most abundant sulfate transporter mRNAs, which increased by ≈2-fold at E4.5 or E6.5, whereas lower levels of Slc26a2, Slc26a6, and Slc26a7 mRNA increased by ≈3- to 6-fold from E4.5. Ileal sulfate transporter mRNA levels were not increased in gestation, but slight decreases (by ≈30-40%) were found for Slc26a3 and Slc26a6. In placentae, Slc13a4 and Slc26a2 mRNAs were most abundant, with levels increasing from E10.5 and peaking (≈8-fold) from E14.5 to E18.5, whereas Slc26a1 increased by ≈3-fold at E18.5. The spatial expression of placental mRNAs was determined by in situ hybridization showing Slc13a4 and Slc26a6 in yolk sac, Slc26a1 in spongiotrophoblasts, and Slc13a4, Slc26a2, Slc26a3, and Slc26a7 in the labyrinthine layer. Within the labyrinth, cell-specific staining revealed Slc13a4 expression in syncytiotrophoblast-II (SynT-II) and Slc26a2 in SynT-I. Together, these data show kidney Slc13a1 and Slc26a1 and placental Slc13a4 and Slc26a2 to be the most abundant sulfate transporter mRNAs in mouse gestation, which likely play important physiological roles in maintaining high maternal serum sulfate levels during pregnancy and mediating sulfate supply to the fetus.

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