Loss of MicroRNA Leads to Abnormal Function and Development of the Adult Mouse Uterus.

Abstract

MicroRNAs (miRNA) are 19-22 nucleotide long, non-coding RNA molecules that post-transcriptionally regulate gene expression by base-pairing with the 3′untranslated region of complementary messenger RNA targets. To date, over 700 miRNAs have been identified in animal cells and have been reported to play a role in numerous vital processes, including: embryonic development, cellular differentiation and proliferation, and apoptosis. Our laboratory recently demonstrated that conditional deletion of Dicer in ovarian granulosa cells and derivatives of the Mullerian duct (i.e. oviduct, uterus, and cervix) by Cre/loxP recombination (Dicerfl/fl;Amhr2-Cre, Dicer-cKO) led to female infertility. This was shown to be the result of a severe oviductal developmental defect that prevented fertilized embryos from entering the uterus. While the uterus of Dicer-cKO females was also disrupted, as evidenced by shortened uterine horns and reduced numbers of uterine glands, its functional ability to establish and maintain pregnancy is not yet known. We first evaluated whether the uterus of pseudopregnant mice could undergo a decidual reaction. To test uterine decidualization, sesame oil was injected into one uterine horn of 3.5 day pseudopregnant mice, while the contralateral horn served as the non-injected control. We next completed an embryo transfer experiment, where 5 blastocysts collected from naturally mated CD-1 mice on day 3.5 (day 0.5 = plug) were transferred into each uterine horn of day 2.5 pseudopregnant adult female Dicer-cKO (n=13) and wild-type mice (Dicerfl/fl or Dicerfl/wt littermates lacking the Amhr2-Cre allele, n=7). Recipient females were then sacrificed on day 7 of pregnancy and the number of implantation sites per mouse recorded. Results of the decidualization experiment indicated that uteri of Dicer-cKO mice (n=11) failed to exhibit a normal decidualization response when compared to wild-type mice (n=7), which exhibited a 3.6-fold increase (p<0.05) in uterine wet weight (mg) after body weight (g) correction. The uteri were 11.8 ± 4.3 mg and 19.7 ± 3.8 mg for the non-injected and 15.0 ± 6.1 mg and 70.8 ± 28.0 mg for sesame oil injected, for the Dicer cKO and wild-type mice, respectively. In the embryo transfer experiment, implantation occurred in 4 of 13 Dicer-cKO recipient females. Interestingly, all implantation sites in the pregnant Dicer-cKO mice were located at the cervical end of the uterine horns. Implantation sites in Dicer-cKO mice appeared less vascular, as evidenced by a blanched color in comparison to the dark red implantation sites observed in wild-type mice. Embryos failed to space appropriately in the two Dicer-cKO recipients with multiple embryos. Histological analysis, however, indicated that the uteri of the pregnant Dicer-cKO mice were able to mount what appeared to be a normal decidualization response with normal embryonic development. We next examined adult female Dicer-cKO uterine tissue histology at 60, 180, 230, and 330 days of age. Uteri from Dicer-cKO mice exhibited adenomyosis, a condition of abnormal endometrial growth within the myometrial layer, which progressively worsened as these animals aged. In conclusion, uterine function in Dicer-cKO mice was compromised and miRNA mediated post-transcriptional gene regulation appears to play a significant role in normal uterine development and function. (poster)