Steroids, peptides and growth factors have all been implicated in promoting spermatogenesis in the vertebrates [1-3]. These molecules may act directly on spermatogonial stem cells (SSC) or their effects may be mediated through somatic cells in the testis, which then generate active mitogens [4-11]. The environmental and resulting molecular mechanisms which regulate spermatogenesis in animals with seasonal reproduction are poorly understood even for mammals.

Little evidence exists linking gene expression by SSC to specific mitogens during the G1- > mitotic S-phase transition. In somatic cells, a common denominator in both steroid and protein based mitogenic pathways is expression of the transcription factor, c-myc [12]. The c-mycprotein heterodimerizes with its protein partner, Max, binds to the E-box sequence (CACGTG) and promotes the expression of target genes relevant to the cell cycle; such as, ornithine decarboxylase [13], elF-4E and 2a [14], cyclin D1 [15], CDC25 phosphatase [16] and p53 [17]. Consequently, expression of c-mycis likely to be an early, detectible response of SSC to mitogens. While studies in vertebrates have demonstrated the effects of various mitogens on c-mycexpression in undifferentiated spermatogonia, the detailed molecular mechanisms involved are unresolved [18, 19].

The aspermatogenic phase of the reproductive cycle of the sea star, Asterias vulgaris,which occurs during the summer, provides an ideal time to test the response of amitotic SSC to treatment with potential mitogens. Simultaneous in vivoandin vitrostudies can be conducted [20-26] and cDNA and antibody probes specific for c-mycin A. vulgarisare available [20].

During the normal reproductive cycle of the sea star, A. vulgaris,SSC alternate between amitotic and mitotic phases. Only amitotic SSC occupy the germinal epithelium from April to August, when testicular c-mycexpression is very low [20]. SSC mitosis is initiated by Fall (September) photoperiod [21] as evidenced by cytology [22], by an increase in the specific activity of ornithine decarboxylase (ODC) [23], by increased concentrations of polyamines (putrescine, spermine and spermidine) [24] and steroids (estradiol, testosterone and 11 ketotestosterone) [25], by increased uptake of 3H-thymidine [26] and finally by substantially increased expression of the c-mycprotooncogene [22, 26].

As spermatogonial mitosis generates primary spermatocytes, cylindrical spermatogenic columns grow towards the lumen of each testis [22]. Centrally, spermatogenic columns include somatic cells analogous to vertebrate Sertoli cells [22, 27 - 28]. These somatic cells are surrounded basally by mitotic SSC and along most of their height by primary spermatocytes that undergo premeiotic S-phase and enter meiotic prophase as they move luminally. After secondary spermatocytes are released from the tips of mature spermatogenic columns, spermatogenesis is completed in the testicular lumen.

A c-myccDNA homolog (pAv-myc -Genbank - 93026376) has been identified for the sea star, A. vulgaris,which encodes a protein with 41% amino acid identity and 46% amino acid conservation to human c-myc[20]. Functional domains of higher conservation include the tripartite transcriptional activation (80%), MAP kinase phosphorylation (100%), nuclear targeting (40%), basic DNA-binding (amino terminal-33%, carboxy terminal-100%), helix-loop-helix (80%) and leucine zipper domains (48%) of human c-myc. When pAv-myccDNA is used as a probe in Northern analysis, it is 200 fold more sensitive than 3H-thymidine incorporation as an indicator of mitosis in testicular cells [26].

In the current study, sea star testes were used to test the hypotheses: 1) That exposure of whole sea stars, containing amitotic testes, to September photoperiod results in premature initiation of SSC mitosis in vivoand 2) That exposure of explanted amitotic testes to 11-ketotestosterone results in the premature initiation of SSC mitosis in vitro. Evidence for c-mycexpression was derived from Northern and Western analyses using authentic probes from A. vulgaris.------