The importance of dietary retinol (vitamin A) and retinoid signaling for normal development and differentiation in the testis has been recognized for many years. Signaling is effected in part through the retinoic acid receptors (RARs), of which there are three isoforms, ?, , and ?. We and others have shown the importance of signaling via the RAR? receptor in particular during spermatogenesis by gene targeting: mice deficient in RAR? (Rara-/-) are viable but the males are sterile, exhibiting defects in the seminiferous epithelium resembling those seen VAD diet. We have also shown that mice treated with an orally bioavailable pan-RAR antagonist become sterile, with similar testicular abnormalities. We have further shown that this induced sterility is reversible even with daily treatments for as long as 4 months upon cessation of drug treatment. Importantly, no detectable side effects were observed at these low doses and normal progeny were sired by fathers after restoration of fertility. This suggested that RAR-antagonists have potential as novel, non-steroidal compounds for male contraception. We propose to pursue this potential as outlined in the following specific aims.
Specific Aim 1. Given the successful use of the pan-RAR antagonist compound 9 to induce male sterility and the recognition that it is RAR? that is critical for regulating spermatogenesis, we propose to test RAR?-selective antagonists that are immediately available and are being developed by our medicinal chemist collaborators in inhibiting spermatogenesis.
Specific Aim 2. RAR? functions as a transcription factor, activating or repressing downstream target genes and antagonists function to block this transcriptional regulation. We therefore propose that such downstream target genes may themselves represent targets for interfering with spermatogenesis and inducing sterility, particularly if they are either testis-specific or function in a testisspecific manner. As spermiation appears to be a cellular process that is exquisitely sensitive to modulation of retinoid signaling, we will therefore focus on elucidating the mechanisms responsible for the improper anchorage of spermatids relative to the basal compartment, failure to translocate from basal compartment to the tubular lumen, and failure to disengage for spermiation, etc. at the cellular and molecular levels. These experiments will involve examining the expression of candidate genes known to be important in these processes and the identification of new genes by state-of-the-art RNA-Seq analysis.
Specific Aim 3. To pursue RAR-antagonists ultimately as contraceptives in men, we propose to undertake a small scale trial in a non-human primate model (the common marmoset). The goal is to set the stage for future pre-clinical trials by examining the efficacy of antagonists with regard to induction of sterility and restoration of fertility. These studies will be done in collaboration with scientistsat the Southwest National Primate Research Center in San Antonio. Our prediction is that the pan-antagonist compound 9 will inhibit spermatogenesis in a reversible manner at doses that will not induce undesirable side effects.
The importance of dietary retinol (vitamin A) and subsequent signaling via its active metabolite all trans retinoic acid (ATRA) for spermatogenesis has been known for many years. ATRA signaling via the nuclear transcription factor retinoic acid receptor alpha (RARa) in particular is essential for spermatogenesis, as demonstrated by gene targeting studies in our lab and by others. Further, we have recently shown that oral administration of a pan-RAR antagonist can induce sterility in male mice, importantly in a reversible manner upon cessation of treatment. These findings support the possibility of the use of retinoid antagonists as a novel, non-steroid hormonal approach to male contraception.
