MicroRNA in the ovary and female reproductive tract.

The ability to enhance reproductive efficiency or inhibit reproductive activity is dependent upon a solid understanding of reproductive physiology. In the female, reproductive success relies on the coordinated actions of the hypothalamic- pituitary-gonadal axis and its regulation of the cervical, uterine, and oviductal tissues. The reproductive tract responds to the cyclical changes in pituitary and ovarian hormones to provide an optimal environment for gamete transport and development, a suitable site for implantation and pregnancy, and protection against pathogens. Whereas much is known about the effect the pituitary and gonadal hormones have on reproductive function, there still remain many unanswered questions concerning the molecular mechanisms related to how these hormones elicit their action(s). The past forty years have shed considerable light on transcriptional gene regulation and cellular signaling pathways within reproductive tissues. However, much less is known about post-transcriptional gene regulation in these tissues, even though it plays an essential role in the synthesis of all proteins and is known to be important in the general regulation of cell differentiation and proliferation. Post-transcriptional gene regulation includes the splicing, editing, transport, storage, turnover, and translation of mRNA, and can result in a difference in expression levels between the transcriptome (mRNA) and the proteome (proteins) of the cell ( Glisovic et al., 2008 ; Gygi et al., 1999 ; Ideker et al., 2001 ). All mRNA transcripts pass through some type of post-transcriptional processing, and although some post-transcriptional gene regulation may be considered almost constitutive in manner, other forms are dynamically regulated ( Hammarskjold, 2001 ; Medina et al., 2008 ). Therefore, it is easy to envision that post-transcriptional gene regulation may be important for coordinating the changes in gene expression that are necessary for the rapid phenotypic changes that occur in the reproductive tract of cycling and pregnant females. In the female reproductive system several examples of post-transcriptional gene regulation have been shown to be involved in gonadal and reproductive tract function. In ovarian granulosa cells, degradation of the LH receptor transcript is increased after the LH surge, and this is mediated by mevalonate kinase, an enzyme in the cholesterol biosynthetic pathway, that also acts as an RNA binding protein ( Wang and Menon, 2005 ). Connexin43, a major component of granulosa cell and oocyte gap junctions, is also translationally repressed in granulosa cells after the LH surge, although the mechanism(s) that mediates this repression has not been identified ( Kalma et al., 2004 ). Additionally, in the ovine uterus, estrogen treatment caused the RNA-binding protein AUF1p45 to stabilize the expression of estrogen receptor α ( Ing et al., 2008 ). Because of the difficulties in examining post-transcriptional gene regulation, few examples exist in reproductive tissues; however, the large numbers of expressed RNA-binding proteins and now microRNA ( miRNA ) suggests this form of gene regulation may play an important role in reproductive tissues ( Berezikov et al., 2006 ; Keene, 2001 ) The recent identification of miRNA as important post-transcriptional gene regulators has led to an explosion in our knowledge of the role post-transcriptional gene regulation plays in organ function. MicroRNA are ~21nt RNA molecules that bind to the 3′UTR of target mRNA to affect their translation. It is predicted that 1–5% of genes encode for miRNA, and they regulate the expression of as many as 30% of mRNA ( Berezikov et al., 2005 ; Lewis et al., 2005 ). Thousands of miRNA have now been identified in a multitude of organisms (microRNA.sanger.ac.uk/). However, the functional importance of individual miRNA and the identity of their specific mRNA targets are just now beginning to be elucidated in rodents and human cell lines. It remains to be determined if the function of miRNA is conserved across species in the same manner as protein-encoding genes. MicroRNA have been shown to be important in many biological processes, including cell proliferation, differentiation, and apoptosis ( Asangani et al., 2008 ; Cloonan et al., 2008 ; Silber et al., 2008 ). Because miRNA have been demonstrated to be important in other developmental and differentiation systems, we propose that miRNA also play a vital role in the development and function of reproductive tissues. In this review we will discuss the current findings that support this hypothesis for the uterus, oviduct, ovary, and oocyte/early embryo and how the dysregulation of miRNA may lead to reproductive disease.

Since miRNA play such a vital role in cell differentiation events, it is easy to envision how the dysregulation of miRNA expression could lead to a disease state. MicroRNA have been demonstrated to be important in multiple types of cancer including ovarian, endometrial, and cervical; as well as non-malignant pathologies, such as uterine fibroids and endometriosis. In fact, miRNA have arguably been more thoroughly studied in the disease state of reproductive tissues than the normal state, and here we will briefly touch on some recent findings in respect to miRNA and their dysregulation in diseased reproductive tissues of women. Ovarian cancer is the sixth most common cancer in women, and miRNA expression has been studied in several types of ovarian cancer and a variety of cancer cell lines ( Corney et al., 2007 ; Dahiya et al., 2008 ; Iorio et al., 2007 ; Nam et al., 2008 ; Yang et al., 2008 ; Zhang et al., 2008 ). To date, the predominant work focuses on elucidation of miRNA signatures that can be used for diagnosis. These studies compare cancerous tissue to either normal tissue or to immortalized cancer cell lines. Interestingly, a set of common miRNA associated with ovarian cancer has not been generated, which may be due to the nature of the controls or reference samples used to compare the cancer tissues. In the uterus, endometrial carcinogenesis affects almost 40,000 women per year. Boren et al. examined the expression of miRNA in endometrial adenocarcinomas versus normal endometrial tissue and identified thirteen miRNA as differentially expressed ( Boren et al., 2008 ). They also identified mRNA differences between the two tissues and found that 9% of the differentially expressed mRNA were predicted targets of the changed miRNA. Uterine leiomyomas (i.e., fibroids) are a benign uterine pathology that affect 30 to 50% of women. Several studies have examined the expression of miRNA in uterine leiomyomas. In two studies, miRNA microarrays were performed to examine the differential miRNA profiles of uterine leiomyomas and normal myometrium. Marsh et al. identified 81 miRNA as differentially expressed, including increased expression of an anti-apoptotic miRNA, miR-21, and decreased expression of a miRNA previously shown to be decreased in pancreatic adenocarcinoma, miR-139 ( Marsh et al., 2008 ). Wang et al identified 45 miRNA as differentially expressed and further grouped the miRNA expression profiles from the uterine leiomyomas based on tumor size and demographics ( Wang et al., 2007 ). Members of the let-7 miRNA family showed high expression in small leiomyomas, but low expression in large leiomyomas ( Wang et al., 2007 ). These miRNA target high-mobility group A2 ( HMGA2 ), a transcription factor highly expressed in large leiomyomas that positively regulates cell growth and proliferation and negatively regulates apoptosis ( Peng et al., 2008 ). Members of the let-7 miRNA family were shown in vitro to prevent HMGA2 expression, and therefore high levels of let-7 miRNA can prevent HMGA2 expression and thus tumor growth. Endometriosis, a disease characterized by the presence of endometrial tissue outside the uterine cavity, causes infertility in 30–40% of patients with the condition. Pan et al. performed a miRNA microarray on human samples of normal endometrium, eutopic endometrium, and ectopic endometrium ( Pan et al., 2007 ). Forty-eight miRNA were identified as differentially expressed. MiR-18a, miR-181a and miR-142-5p were shown to have predicted target transcripts that were already implicated in endometriosis including estrogen receptor alpha, estrogen receptor beta, and progesterone receptor, respectively. Moreover, the expression of several miRNA (miR-20a, miR-21, and miR-26a) was regulated by treatment with the ovarian steroids estrogen or progesterone (medroxyprogesterone acetate), as well as the steroid receptor antagonists ICI-182780 and RU486 ( Pan et al., 2007 ).

The role of miRNA in reproductive tissue function and development are presently being elucidated. To date much of the work has focused on miRNA profiling of the reproductive organs, but the expression, regulation, and function of miRNA within specific tissues and cells still needs to be determined. To help establish functional connections, conditional Dicer knockout mice have been used to show the consequences that the lack of miRNA have on ovarian, oviductal, uterine, and oocyte/embryo function and development. Much less is known about the specific miRNA and their targets that cause these phenotypic effects, but this area of research is rapidly moving forward. Within the next several years it is expected that a wealth of information regarding miRNA-mediated post-transcriptional gene regulation in reproductive tissues will be known. Because of the importance miRNA have in a variety of tissues and in diseases, understanding the role of miRNA in reproductive tissues will provide insight into how we can better enhance reproductive efficiency or inhibit reproductive activity.