Even after decades of research, the molecular basis for why spermatogenesis requires testosterone is not completely understood. This is in part because very few androgen-regulated genes have been definitively identified so far. It is likely that most androgen-regulated genes in the testis respond indirectly to androgen via activation or suppression of transcription by other trans-acting factors directly regulated by androgen. Recently, we have shown that one class of such androgen-regulated trans-acting factors in the testis are microRNAs (miRNAs), small non-coding RNA molecules that inhibit the translation of the target transcript by binding to complementary cis-regulatory sites in the mRNA. Specifically, we have shown that a number of these miRNAs are upregulated (suggesting their gene targets would be downregulated) in the absence of androgen. Our long term goal is to understand the in vivo function and mechanism by which these androgen-responsive miRNAs regulate critical pathways essential for male fertility. Toward this goal, we generated transgenic mice expressing miR-471 driven by the Sertoli cell-specific promoter Rhox5Pp that we have previously extensively characterized. Our preliminary analyses of miR-471 transgenic mice reveal that Sertoli cell-specific overexpression of miR-471 resulted in dramatically increased germ cell apoptosis, multinucleated giant cells suggesting abortive meiosis in spermatocytes, impaired postmeiotic germ cell differentiation and extensive germ cell sloughing, a phenotype that correlates with impaired Sertoli cell-Sertoli cell/germ cell adhesion at the blood-test barrier, which is an androgen-dependent event during spermatogenesis. These findings led us to hypothesize that by targeting specific transcripts, miR-471 expression in the Sertoli cells regulates specific androgen-dependent steps critical for germ cell development and progression.
Two specific aims are proposed to test our hypothesis:
In aim 1, we will extend our preliminary analysis and further characterize miR-471 transgenic mice. We will specifically assess the integrity of Sertoli cell-Sertoli cell/germ cell adhesion as well as germ cell meiotic arrest using molecular and biochemical approaches. This study will be highly significant as miR-471 transgenic mice represent the first genetic model to study the function of specific miRNAs in the Sertoli cells and miR-471 will be the first proven androgen-regulated miRNA important for male fertility.
In aim 2, we will validate miR-471 gene targets and address their role in mediating Sertoli cell-specific function of miR-471 using in vitro and ex-vivo approaches. The androgen-regulated miRNA on which this proposal is focused provide an opportunity to begin to address this. The proposed work will provide important leads to clearly understand the molecular mechanism by which androgen regulates critical pathways essential for male fertility. Furthermore, this research initiative may provide insights into the putative role of miRNA in androgen related diseases such as prostate cancer and androgen insufficiency syndrome.
The proposed work will provide important leads to understand the molecular mechanism by which androgen regulates critical pathways essential for male fertility. In addition, since a large number of genes are regulated by miRNAs, and miRNA levels can be easily manipulated using synthetic oligoribonucleotides, some of these discoveries may translate into novel therapies for infertility and/or new approaches to male contraception. Furthermore, this research initiative will provide insights into the putative role of miRNA in androgen related diseases such as prostate cancer and androgen insufficiency syndrome.