The process of mammalian sex determination and differentiation is of fundamental importance and remains one of the basic questions of human biology. Recently, strong genetic evidence has determined the piece of the Y-chromosome thought responsible for testes determination. with the discovery of the SRY gene, Sex-determining Region of Y, the beginning of this gene cascade may be defined, and the search for downstream target genes can begin. While the exact identification of downstream targets may prove difficult, experimental evidence suggest that the evolution of the Sertoli cell-type of the testis is intimately linked to the Y- chromosome. As the """"""""indifferent"""""""" bipotential genital ridge begins to differentiate into testes, one can visualize the Sertoli cells aligning themselves into cord-like structures. Shortly after, a critical function of the Sertoli cell will be the expression and local release of the hormone, Mullerian inhibiting substance, MIS. During a critical time, the secretion of MIS results in the regression or """"""""cell death"""""""" of the Mullerian duct, the anlagen of internal female genitalia. Thus, regulation of MIS gene expression is directly coupled to the differentiation of the Sertoli cell and the execution of the Y- chromosomal developmental programs. Oddly enough, MIS is also expressed at adult granulosa cell of the ovary; functions in this """"""""sister"""""""" cell type of the Sertoli cell remain unclear. Here, it is proposed to define and isolate the developmental regulator(s) of MIS gene expression. Preliminary data suggest that a conserved element, MIS-RE-1, close to the promoter of the mouse MIS gene is bound by a Sertoli-cell specific protein. Further, point mutations that abolish binding also decrease transcription activity as measured in transfection assays using primary Sertoli cells. Binding activity of this protein is roughly correlated to the time of peak MIS transcript expression. Moreover, the same MIS- RE-1 element is also bound by an ovarian-specific protein exhibiting distinct binding specificity and a relative molecular weight different from that observed in the testes.
Aim #1 will characterize the mouse MIS gene by defining the transcriptional start site and comparing its sequence with that of the human MIS gene. Standard biochemical analyses and functional studies will probe the protein-DNA interactions between the MIS-RE-1 and the testis-specific MIS-REBP (binding protein). Complete ontogeny of testes-MIS-REBP activity and ovary-specific activity will be undertaken.
Aim #2 proposes to purify and clone the Sertoli- specific protein binding to the MIS-RE-1 site. The purification and cloning of the testes-MIS-REBP will follow well-established protocols for DNA-binding proteins. If the testes-MIS-REBP acts to initiate MIS-gene expression its transcript and protein expression should correlate with the onset of MIS gene activation. This hypothesis can be tested following isolation of the MIS-REBP cDNA clone.
Aim #3 will attempt to characterize the developmental effects of MIS cis-elements by a simple set of transgenic mouse experiments. In choosing to study the initial activation of MIS gene expression, it is likely that these studies will shed light on basic molecular mechanisms underlying the development and maturation of reproductive endocrine organs.
|Ikeda, Y; Shen, W H; Ingraham, H A et al. (1994) Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654-62|