Small molecule Hedgehog pathway antagonists.

Leveraging our quinolone-1-(2H)-one based Hedgehog signalling pathway (HSP) inhibitors we have developed two new classes of HSP inhibitors based on: l-tryptophan and benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amine. Synthesis of focused compound libraries identified six l-tryptophan based inhibitors, and two stimulators, of Gli at 10 µM compound concentration. 2,4-Dichloro-13 and indole 16 suppressed mRNA expression of Ptch1 in Shh LIGHT2 cells, with 13 suppressing and 16 stimulating Gli2 mRNA expression. Focused library development of the benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amine scaffold afforded two sub-micro molar potent inhibitors of Gli expression with 5-methoxy-1H-indole-2-carboxylic acid benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amide 29 and 5-chloro-1H-indole-2-carboxylic acid benzo[1,3]dioxol-5-ylmethyl-[2-(1H-indol-3-yl)-ethyl]-amide 30 returning IC50 values of 0.5 and 0.24 µM, respectively. Neither 29 nor 30 acted directly on Smo with our data supporting inhibition of the HSP downstream of Smo.

Developmental expression of Musashi-1 and Musashi-2 RNA-binding proteins during spermatogenesis: analysis of the deleterious effects of dysregulated expression.

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

Dynamin 2 is essential for mammalian spermatogenesis.

The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.