Role of Dicer in female fertility.

Oocyte fertilization and early pre-implantation embryonic development occurs within the oviduct. Subsequently, the developing embryo passes through the uterotubal junction and enters the uterus where implantation, placentation, and embryonic/fetal development occur. In addition to granulosa cells, Amhr2-Cre is expressed in the oviduct, uterus, cervix, and anterior portion of the vagina [ 39 ]. Loss of Dicer in mouse oviducts results in a dramatic phenotype consisting of shortened tubule length, loss of oviductal coils, and large fluid filled sacs [ 35 – 38 ]. Histological analysis of oviducts from Dicer Amhr2-cKO females revealed loss of the smooth muscle layer and disorganization of the epithelium in the oviduct, particularly in the isthmus (region near the uterus) of the oviduct [ 35 – 37 ]. Collection of embryos on day 4 post-mating found all embryos retained in the oviduct of Dicer Amhr2-cKO females, whereas embryos in wild-type females had all traversed the uterotubal junction and resided within the uterus [ 35 ]. Similarly, affinity chromatography beads similar in size to preimplantation embryos that were injected into oviducts were unable to enter the uterus in Dicer Amhr2-cKO mice [ 37 ]. Since ovulated Dicer Amhr2-cKO oocytes were fertilized following mating [ 35 ], this would argue that the block is unidirectional, and/or due to an effect on smooth muscle contractility, cilia, or oocyte size that prevents transport. Nagaraja et al. [ 36 ] identified 28 down-regulated miRNAs in oviducts from the Dicer Amhr2-cKO mouse model, 23 of which were predicted to target at least one member of the Wnt or Hox family of genes [ 36 ]. In fact, the defect in oviductal transport of the Dicer Amhr2-cKO mouse phenocopies that seen in mice deficient in Wnt/β-catenin signaling [ 48 , 49 ]. Indeed, β-catenin levels were reduced in oviducts and uteri of Dicer Amhr2-cKO females [ 35 ]. In addition to developmental effects, it appears that loss of miRNA in the oviduct might also influence factors secreted into the oviductal lumen, as embryos collected three days after mating from the oviducts of Dicer Amhr2-cKO females were developmentally delayed compared to those collected from wild-type animals [ 35 ]. Similar to the oviductal phenotype, the uteri of Dicer Amhr2-cKO animals were also developmentally compromised (with 3 of 4 reports noting severe defects [ 35 – 37 ]). The length and diameter, as well as weights of uteri collected from eCG-stimulated juvenile Dicer Amhr2-cKO females were smaller than wild-type littermates [ 35 , 36 ]. Histological analysis revealed the presence of all tissue layers within the uterus [ 35 – 37 ], although reduced numbers of uterine glands and a thinner myometrial layer was observed in the one Dicer Amhr2-cKO mouse model [ 35 , 37 ]. Gonzalez et al [ 37 ] further noted that the uterine glands reside in close proximity to the myometrial layer, mimicking a human condition referred to as adenomyosis [ 50 ]. The uteri of these mice (n=3 cKO) were not able to sustain pregnancy following embryo transfer. Interestingly, histological analysis failed to observe any defect in other Dicer floxed mouse lines [ 36 ], and adenogenesis (i.e., gland formation and location), estrogen responsiveness, and stimulus-induced decidualization reaction all appeared normal [ 36 ]. Expression of Dicer mRNA and protein as well as the Argonaute proteins is abundant in the mouse uterus on days 4 through 8 of pregnancy (day 1=presence of vaginal plug), suggesting that miRNA synthesis is ongoing [ 51 ]. Furthermore, microarray analysis of miRNA present in the uterus on day 4 of pregnancy (receptive phase) and at implantation sites suggests that miRNAs are important for establishing pregnancy [ 51 , 52 ]. Comparison of miRNA expression within uterine tissues collected on days 1 and 4 of pregnancy identified 32 miRNA upregulated on day 4 [ 53 ]. Expression of miR-101 and miR-199* were stimulated by estradiol and found to post-transcriptionally regulate prostaglandin synthase-2, an enzyme necessary for implantation in mice [ 53 ]. Further analysis in the pregnant uterus found 13 miRNAs upregulated in implantation sites compared to inter-implantation sites [ 53 ]. Analysis of miR-21, one of the upregulated miRNAs, found that expression was dependent upon the presence of an activated embryo [ 53 ]. Uterine miRNAs also appear to be regulated by estrogen, as 49 miRNAs were found to be differentially regulated in response to estradiol [ 54 ]. Taken together, these studies suggest a role for uterine miRNAs in implantation and pregnancy.

To date, the study of Dicer function in female reproductive tissues is limited to a few reports ( Figure 2 ). However, these studies all clearly demonstrate that Dicer and its enzymatic products are crucial for female fertility. Conditional deletion of Dicer in the oocyte, ovary, oviduct, and uterus provide convincing evidence that miRNAs/siRNAs are necessary for the overall development and function of the female reproductive system. Recent studies demonstrated that expression of Amhr2-Cre recombinase was leaky, with recombination also occurring in the brain, pituitary, heart, and tail in addition to the known tissues expressing Amhr2 [ 38 , 55 ]. Additional studies using more refined temporal and cell specific Cre-recombinases are needed to further our understanding of post-transcriptional gene regulation in the female reproductive system ( Figure 2 ). Identification of individual miRNAs or siRNAs in each female reproductive organ will allow researchers to better understand the mechanisms of gene regulation that allow for successful reproduction. Thus far, little is known about the role of siRNAs in somatic cells of the reproductive system, although they appear to be highly abundant [ 13 , 14 ]. Additional research is needed to determine the functional role siRNA molecules exert on tissue and organ function. Recently, antagomirs and locked nucleic acid oligonucleotides have been used to downregulate specific miRNA expression in a variety of cells in vitro and tissues in vivo [ 56 , 57 ]. It is anticipated that establishing the spatiotemporal expression patterns of miRNA in the female reproductive tract will provide targets for drug and therapeutic treatments for reproductive diseases such as endometriosis, uterine leiomyomas, and ovarian, uterine, and cervical cancers. Moreover, understanding the role that post-transcriptional gene regulation plays in reproduction will facilitate the elucidation of the etiologies leading to reproductive failure and hopefully provide methods/targets for treatment of infertility and provide new means of contraception.

Regulation of fertility in the female is a dynamic and highly regulated process that requires the coordinated actions of multiple tissues and organ systems (e.g. hypothalamus, pituitary, ovary, and reproductive tract) to develop a fertilizable gamete as well as provide a suitable environment for fertilization and subsequent fetal development. To attain this optimal environment, the female reproductive system must be highly malleable to subtle changes in hormones and other external cues. A large body of evidence supports a role for transcriptional regulation in mediating these changes, and recent evidence suggests a hereto underappreciated role for post-transcriptional gene regulation in reproductive tissue and organ function [ 1 ]. Post-transcriptional gene regulation encompasses all aspects of messenger RNA (mRNA) turnover, processing, storage and translation, and provides cells with additional mechanisms to regulate protein content following transcription events. Recently, study of post-transcriptional gene regulation has surged due to the discovery of small non-coding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) [ 2 ]. Incorporation of miRNA and siRNA into RNA-induced silencing complexes (RISC) allows for the targeting of specific mRNA transcripts, ultimately providing cells with a post-transcriptional regulatory mechanism to either induce or inhibit protein production in response to stimuli, independent of commencement or cessation of mRNA transcription (for reviews, see [ 3 , 4 ]). This review focuses on the RNA endonuclease III (RNAse III), Dicer, and its enzymatic products, miRNA and siRNA, which elicit post-transcriptional regulatory responses. Dicer is a cytosolic multi-domain protein comprised of a RNA helicase, domain of unknown function (DUF283), Piwi Argonaute Zwille (PAZ) domain, two RNAse III domains, and a double stranded RNA binding domain (dsRBD) [ 5 , 6 ]. The RNA helicase unwinds long dsRNA precursors, whereas the dsRBD and PAZ domains are essential for Dicer binding to dsRNA and for determining the length of the siRNA or miRNA products, respectively. Cleavage of miRNA or siRNA precursors is dependent on the two RNAse III domains within the Dicer protein. Dicer is essential in miRNA and siRNA biogenesis ( Figure 1 ), and its function is critical to the cell, as general knock-out of Dicer1 (hereafter referred to as Dicer) in the mouse causes morphologic abnormalities and stunted growth in embryonic day (E) 7.5 embryos and lethality by E11.5 [ 7 ]. To further explore Dicer function, multiple groups developed Dicer alleles with the second RNAse III domain flanked by loxP sites ( Dicer fl/fl ) to facilitate conditional knock-down (cKO) of Dicer fl/fl via tissue specific Cre recombination [ 8 – 12 ]. Although miRNAs are abundant in mammalian somatic tissues and thus most affected by the loss of Dicer, several reports using deep sequencing methods have suggested that siRNAs also might play important role(s) in both somatic tissues and germ cells [ 13 , 14 ]. Therefore, whereas loss of Dicer within somatic tissues has typically been linked to altered miRNA biogenesis, changes observed in conditional Dicer fl/fl knockout mice might be due to a combined loss of miRNA and siRNA. MicroRNAs are derived from highly conserved genes that bind partially complementary sequences in the 3′-untranslated region (3′UTR) and/or coding regions of target mRNA transcripts to regulate gene expression ( Figure 1 , for review [ 3 , 4 ]). To date, 706 and 547 miRNAs have been identified in the human and mouse, respectively (Version 13 Sanger MirBase) [ 15 ]. Because each miRNA is derived from a specific pre-miRNA hairpin loop and represents a specific gene product, a standardized nomenclature for mammalian miRNAs has been established ( Box 1 ). The functions of miRNAs are diverse and play a role in numerous processes including cellular proliferation and differentiation, embryonic development, and apoptosis (for review, see [ 16 – 18 ]). To provide structure in naming and characterizing the thousands of recently identified miRNAs, the Sanger Institute miRNA Registry was established ( http://microrna.sanger.ac.uk/ ; [ 15 ]. The current nomenclature for animal miRNAs is described below. The names of miRNAs consist of four components, each conveying a specialized piece of information about the given miRNA: species, form (precursor or mature), identification number, and origin of miRNA (either processing origin or chromosomal origin). Each component is separated by dashes and is represented by the following template: xxx-miR-#-suffix. The xxx signifies the species (i.e., hsa=human, mmu=mouse). To distinguish between the precursor and mature forms of miRNA, a small case ‘r’ (‘mir’) represents the precursor form of the miRNA, whereas an uppercase ‘R’ (‘miR’) represents the mature form of the miRNA. Generally, an identification number is assigned in sequential order of discovery, thus recently identified miRNA have larger numbers. The suffix identifier (which might or might not be separated by a dash depending on the suffix, Figure I ) denotes either the processing or chromosomal origin of the miRNA. For the processing origin, opposite arms from a single pre-miRNA are denoted ‘5p’ and ‘3p’. After one arm is experimentally identified as the predominant arm, the less predominant arm is labeled with an asterisk suffix. For genomic origin, miRNAs that arise from different genomic loci but have identical mature sequences are labeled with numbered suffixes, and if they arise from paralogous genomic loci and have highly similar mature sequences, they are labeled with lettered suffixes. In a few cases, miRNA genes have been identified at the same chromosomal location and are found on opposing DNA strands (sense versus antisense) and thus have unique mature miRNA sequences. The miRNA on the antisense chromosome is identified with an ‘as’ suffix and on the sense chromosome an ‘s’ suffix. A given miRNA can have several suffixes. For example, hsa-miR-19b-1, which originates from chromosome 13, is identical to hsa-miR-19b-2 which originates from chromosome X, and is paralogous (shares 70% of mature sequence) to hsa-miR-19a which originates from chromosome 13. Nomenclature of miRNA suffixes Exogenous siRNAs injected into cells have been widely used to knock down gene expression (reviewed in [ 19 ]). Endogenous siRNAs are derived from dsRNA (i.e., pseudogenes, transposable elements, or protein-coding genes). Due to the nature of their biogenesis, siRNAs are typically thought to be fully complementary to their target transcripts, and a standardized nomenclature or estimate of the number of mammalian siRNAs has yet to be established ( Figure 1 ). It remains possible that siRNAs associated with RISC complexes could also act like miRNA and not require full complementation to affect post-transcription gene regulation. Endogenous siRNAs derived from pseudogenes have been shown to target specific mRNA transcripts and regulate transposable elements in the mammalian oocyte [ 13 , 14 , 20 ]. The biological significance of siRNA retrotransposon silencing is thus far unique to the oocyte, and its exact role has yet to be defined [ 20 ]. Limited examples of post-transcriptional gene regulation exist within the somatic cells of the reproductive system [ 21 , 22 ]. However, as the function of Dicer and its products (miRNAs and siRNAs) are studied in the female reproductive tract, vital roles for such post-transcriptional gene regulation in female fertility are becoming evident [ 1 ]. In addition, due to the involvement of miRNAs in these crucial biological pathways, it is not surprising that they have been implicated in a number of reproductive diseases and cancers of the reproductive system (for review, see [ 23 – 25 ]). Gaining a better understanding of Dicer generated miRNAs and siRNAs will provide important insight into regulation of the female reproductive system and perhaps the mechanisms that regulate fertility and etiology of reproductive diseases.