The pituitary gland stimulates cycles of ovarian follicle maturation through its production and subsequent release of follicle-stimulating hormone (FSH). FSH is secreted in two """"""""peaks"""""""" across the human menstrual cycle and this pattern of release is governed by endocrine hormones hypothalamic releasing peptides and paracrine growth factors that converge on pituitary gonadotrope cells to stimulate or inhibit FSH production. The projects described in this proposal concern the mechanisms of action of one class of regulatory factors, the activins. Activins, produced within gonadotropes, stimulate the production of one of the two protein subunits that comprise FSH, the FSHbeta subunit. Activins bind to cell-surface receptors that activate intracellular signaling proteins in the SMAD family. Specifically, activins stimulate SMAD2 and SMAD3 phosphorylation and nuclear translocation. Once in the nucleus, SMAD3 stimulates transcription of the FSHbsubunit gene. Early indications were that SMAD2 was not involved in this process; however, it is clear that SMAD3 alone cannot account for activin's stimulation of FSHbeta.
In Specific Aim 1, SMAD2-dependent signal transduction will be antagonized and the resulting effects on activin-stimulated FSHbeta transcription examined. SMAD3 binds a cis-acting regulatory element in the FSHbeta promoter. This site is necessary but not sufficient for activin and SMAD3-stimulated gene expression. Another region in the more distal promoter is also required for the transcriptional response. The position of this regulatory region will be mapped and the transcription factors acting there will be identified. To demonstrate an in vivo role for SMAD3 in FSHa expression, a conditional transgenic mouse model will be produced in Specific Aim 2. Here, the tetracycline-inducible system will be used to express a dominant negative (dn-) form of SMAD3 in gonadotropes of adult mice. The effects of dn-SMAD3 on FSH synthesis and secretion as well as estrous cyclicity and fertility will be examined. Finally, FSHbeta is expressed within gonadotropes, but not in other cell types of the body. The mechanisms controlling this cell-restricted gene expression are not understood. SMAD3, in concert with a zinc-finger transcription factor, GATA-2, stimulates basal FSHbeta transcription in cells that under other circumstances do not express this gene.
In Specific Aim 3, the mechanisms controlling the synergistic effects of GATA-2 and SMAD3 will be elucidated and the necessity for GATA-2 in basal FSHb expression examined. Collectively, the results of these experiments will shed new light on the mechanisms controlling basal and activin-stimulated FSHbeta expression. Given the critical importance of FSH for fertility in women, these results may aid in the design of novel contraceptives or may pinpoint causes of infertility that have thus far eluded our detection.
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