In the Sf1/Cre-mediated Dicer1 KO mice, adrenal cortical cells are the first SF1-positive population that undergoes apoptosis at fetal stages, followed by testicular Leydig cells and Sertoli cells, respectively. Adrenal degeneration in the Dicer1 KO starts between 16.5-18.5 dpc and by the time of birth, adrenal cortical cells are almost completely abolished. In the testis, however, degeneration becomes apparent only after birth and progresses rapidly. Loss of somatic cells in the testes eventually leads to germ cells loss in the Dicer1 KO testis. By P5, almost all of the functional structures and cell types in the testis are no longer present. Intriguingly, ovaries, which derive from the same SF1-positive primordium as adrenal and testis, show no morphological and cellular changes from fetal stages to P5 in response to the loss of Dicer1. Increased apoptosis has been reported in tissues that lack Dicer1 [5–8]. Increased cell death in testes and adrenals in our study further suggests that Dicer1 and microRNAs processed by DICER play a universal role in maintaining cell survival.
In the adrenal, the cortical cells are derived from at least two sources: 1) the SF1-positive adrenal primordium, which forms the foundation of the organ and 2) SF1-negative capsular cells, which contribute to further growth of the adrenocortex [23–26]. It is known that loss of Sf1 leads to apoptosis of adrenal cortical cells and adrenal dysgenesis at birth [14, 27]. Loss of Dicer1 in the SF1-positive cortical cells also leads to apoptosis of the cortex; however, the degeneration process occurs much later than that in the case of Sf1 KO. It is possible that DICER-regulated miRNAs control genes that are critical for cortical cells survival, such as Sf1. However, in the Dicer1 KO adrenal, SF1 is still present in the remaining cortical cells. Depletion or mutation of Cited2, Wt1, and Pbx1 also resulted in prenatal adrenal dysgenesis in mouse embryos [28–30]. Involvement of Dicer and miRNAs in regulation of these genes in adrenal development remains to be determined.
Adrenocortical degeneration was also reported in β-catenin conditional KO mice . β-catenin deficiency mediated by the same Sf1/Cre resulted in underdeveloped adrenal cortex; however, no apoptosis was found at fetal stages. These data suggest that regulation of adrenal development via β-catenin is probably independent from DICER-regulated miRNA machinery or vice versa.
Dicer and other components of the miRNA-mediated interference machinery are present in the testis, including germ cells and Sertoli cells [31–34]. Germ cell-specific Dicer1 KO mice were generated using the TNAP/Cre that is active in the primordial germ cells [9, 10]. Loss of Dicer1 in germ cells led to defects in proliferation and differentiation of spermatogonia and abnormal morphology and motility of sperm [9, 10]. Sertoli cell-specific Dicer1 KO mice was generated using the anti-Müllerian hormone Cre (Amh-cre) line that targets Sertoli cells starting at ~15.5 dpc . Loss of Dicer1 in Sertoli cells resulted in impaired spermatogenic waves and complete absence of spermatozoa. In addition, increased Sertoli cell apoptosis was found at P5 and germ cells underwent apoptosis at P15, eventually leading to complete testis degeneration at P180. In our Sf1/Cre-mediated Dicer1 KO model, we targeted both Sertoli cells and Leydig cells at an earlier stage (10-11 dpc). We observed testicular degeneration at an earlier time point and in a more dramatic fashion than was reported in the Sertoli cell Dicer1 KO. In our model, almost all testicular structures and cell types were absent at P5. As expected, both Sertoli and Leydig cell populations underwent apoptosis albeit within different time frames (the number of Leydig cells decreases first, followed by Sertoli cells). Although male germ cells are negative for SF1, their survival was affected in the somatic cell-specific Dicer1 KO testis. This is expected as Sertoli cells are known to provide structural supports and differentiation cues to support spermatogenesis . Although Leydig cells were also affected in our model, we do not believe that their demise is responsible for the germ cell loss phenotypes based on the fact that male germ cells are not known to respond to androgens, the major product of Leydig cells.
Dicer1 is also expressed in the female reproductive tract and the ovary, including oocytes, theca cells and granulosa cells [36–39]. Many miRNAs are synthesized in ovaries at different stages of folliculogenesis [40–42]. It has been reported that morphogenesis and function of the female reproductive tract were affected in the absence of Dicer1 . To study the in vivo role of Dicer1 in the mouse ovary, granulosa cell specific Dicer1 KO mice were generated by using Amhr2/Cre, which is expressed in granulosa cells of preantral and antral follicles [11, 43–45]. Two different Dicer1-floxed strains were used and both showed that loss of Dicer1 in granulosa cells resulted in decreased ovulation rate, trapped oocytes in luteinized follicles and increased numbers of atretic follicles [11, 18, 46]. Fertilized oocytes collected from granulosa cell-specific Dicer1 KO females had decreased ability to progress to the two-cell stage . We were not able to examine the consequence of loss of Dicer1 on folliculogenesis due to death of Dicer1 KO animals after P5 as a result of adrenal cortex degeneration. However at least at P5, Dicer1 KO ovaries show no signs of degeneration.