Spermatogonia of rainbow trout: III. In vitro study of the proliferative response to extracellular ATP and adenosine

Unlike in higher vertebrates, in fish it is not known whether the nerve sup ply of the testis can influence testicular functions or not, In addition to neurotransmitters, nerve terminals may release ATP and adenosine in the ex tracellular medium. On the assumption that these molecules might be release d by fibers innervating the teleost testis, it is possible that they partic ipate in the control of testicular function and, maybe, in the control of s permatogonia (Go) proliferation. This study addresses this issue. We have i nvestigated the ability for extracellular ATP and adenosine to influence th e in vitro incorporation, either basal or GTH-, IGF-I- and suramin-stimulat ed, of H-3-thymidine (H-3-Tdr) by trout Go. Mixed suspensions of somatic an d germ cells prepared from testes, which were immature or spermatogenetic, were cultured usually for 4.5 days in the presence or not of the tested mol ecules; H-3-Tdr was added during the last day in culture. In our cell cultu re conditions, 25 to 250 mu M adenosine, ATP, ADP, and AMP stimulated the H -3-Tdr incorporation by Go from prespermatogenetic testes and from testes s tarting spermatogenesis, in a dose-dependent way. The effect of these molec ules decreased when the testes were move advanced in spermatogenesis and it became inhibiting when the testes were in mid-spermatogenesis, Five'-N-eth ylcarboxamidoadenosine (NECA) was as potent as adenosine in stimulating or inhibiting H-3-Tdr incorporation, while R-N-6-(2-phenylisopropyl)adenosine (R-PIA) always had a marked inhibiting effect. Adenosine 5'-O-(3-thiotripho sphate) (ATP gamma S; 25-200 mu M), a non-hydrolysable analogue of ATP, whi ch had no effect on Go from prespermatogenetic testes (collected October-Fe bruary) and from testes in advanced spermatogenesis, stimulated H-3-Tdr inc orporation by Go from testes at the beginning of spermatogenesis very effic iently. The order of potency of the different ATP analogues was as follows: ATP gamma S > ATP congruent to alpha,beta-methylene-ATP > UTP > 2-methylth io-ATP. These data suggest that A(2) adenosine receptors and P-2 receptors would be present on unidentified testicular cells. The stimulating effect o f adenosine/ATP was additive with that of either GTH-I or IGF-I or suramin when the cells were from testes at the beginning of spermatogenesis, but ad enosine suppressed their effect when the cells were from testes in mid-sper matogenesis. In conclusion, our results suggest that in the trout extracell ular adenosine and ATP are able to influence the in vitro proliferation of Go, and are potential candidates for mediating the possible influence of th e nervous system on the induction, speeding up, then slowing down of sperma togenesis. (C) 1999 Wiley-Liss, Inc.