Previous work in dogfish, Scyliorhinus canicula, has identified the testicular germinative area as the spermatogonial stem cell niche. In the present study, an in vitro co-culture system of spermatogonia and somatic cells from the germinative area was developed. Long-term maintenance of spermatogonia has been successful, and addition of GDNF has promoted the development of clones of spermatogonia expressing stem cell characteristics such as alkaline phosphatase activity and has allowed maintenance of self-renewal in spermatogonia for at least 5 mo under culture conditions, notably by decreasing cell apoptosis. Furthermore, clones of spermatogonia expressed the receptor of GDNF, GFRalpha1, which is consistent with the effect of GDNF on cells despite the lack of identification of a GDNF sequence in the dogfish's transcriptome. However, a sequence homologous to artemin has been identified, and in silico analysis supports the hypothesis that artemin could replace GDNF in the germinative area in dogfish. This study, as the first report on long-term in vitro maintenance of spermatogonia in a chondrichthyan species, suggests that the GFRalpha1 signaling function in self-renewal of spermatogonial stem cells is probably conserved in gnathostomes.

Aromatase (CYP19) catalyzes the conversion of androgens into estrogens. It is in particular involved in development, reproduction and breast cancer. One of its polymorphisms, W39R localized in the N-terminal region of CYP19, significantly decreases breast cancer risk among Japanese women and was chosen for this study. In this work, we studied the structure-function relationships between W39R polymorphism and CYP19 enzyme activity. To examine the kinetic properties of the mutant W39R recombinant protein in transfected human cells devoid of steroidogenic activity. Expression vectors for the wild-type or the mutated R39 aromatase were transiently transfected into E293 human embryonal kidney cells. The conversions of androstenedione to estrone and of testosterone and nortestosterone to 17beta-estradiol were assayed by RIA. Expression of recombinant cDNAs was analyzed by semi-quantitative RT-PCR and immunoblotting. W39R recombinant protein was devoid of aromatase activity whatever the substrate used. This absence of activity was not due to the lack of expression of the recombinant enzyme since the mRNA and protein were detected. Our present in vitro study shows that the R39 mutant is unable to synthesize estrogens. This work provides a novel observation, being consistent with the fact that Japanese women with the variant allele (arg) have significantly lower risk of developing a breast tumor.

Cancer therapy is currently a major challenge within the research community, especially in reducing the side effects of treatments and to develop new specific strategies against cancers that still have a poor prognosis. In this context, alternative strategies using biotechnologies, such as marine peptides, have been developed based on their promise of effectivity associated with a low toxicity for healthy cells. The purpose of the present paper is to investigate the active mechanism of two peptides that were isolated from the epigonal tissue of the lesser spotted dogfish Scyliorhinus canicula L., identified NFDTDEQALEDVFSKYG (K092A) and EAPPEAAEEDEW (K092B) on the in vitro growth inhibition of ZR-75-1 mammary carcinoma cells and MDA-Pca-2b prostate cancer cells. The effects of the peptides on cell proliferation and cell death mechanisms were studied by the flow cytometry and immunofluorescence microscopy approaches. The results have shown the onset of both K092A- and K092B-induced early cytoskeleton changes, and then cell cycle perturbations followed by non-apoptotic cell death. Moreover, impedance perturbation and plasma membrane perforation in ZR-75-1 K092A-treated cell cultures and autophagy inhibition in MDA-Pca-2b K092B-treated cells have been observed. In conclusion, these two bioactive peptides from dogfish exhibit antineoplastic activity on the human prostate and breast cancer cells in vitro.