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

Abstract

Unlike in higher vertebrates, in fish it is not known whether the nerve supply of the testis can influence testicular functions or not. In addition to neurotransmitters, nerve terminals may release ATP and adenosine in the extracellular medium. On the assumption that these molecules might be released by fibers innervating the teleost testis, it is possible that they participate in the control of testicular function and, maybe, in the control of spermatogonia (Go) proliferation. This study addresses this issue. We have investigated the ability for extracellular ATP and adenosine to influence the in vitro incorporation, either basal or GTH‐, IGF‐I‐ and suramin‐stimulated, of 3H‐thymidine (3H‐Tdr) by trout Go. Mixed suspensions of somatic and 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 molecules; 3H‐Tdr was added during the last day in culture. In our cell culture conditions, 25 to 250 μM adenosine, ATP, ADP, and AMP stimulated the 3H‐Tdr incorporation by Go from prespermatogenetic testes and from testes starting spermatogenesis, in a dose‐dependent way. The effect of these molecules decreased when the testes were more advanced in spermatogenesis and it became inhibiting when the testes were in mid‐spermatogenesis. Five′‐N‐ethylcarboxamidoadenosine (NECA) was as potent as adenosine in stimulating or inhibiting 3H‐Tdr incorporation, while R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) always had a marked inhibiting effect. Adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS; 25–200 μM), a non‐hydrolysable analogue of ATP, which had no effect on Go from prespermatogenetic testes (collected October–February) and from testes in advanced spermatogenesis, stimulated 3H‐Tdr incorporation by Go from testes at the beginning of spermatogenesis very efficiently. The order of potency of the different ATP analogues was as follows: ATPγS > ATP ≅ α,β‐methylene‐ATP > UTP > 2‐methylthio‐ATP. These data suggest that A2 adenosine receptors and P2 receptors would be present on unidentified testicular cells. The stimulating effect of 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 adenosine suppressed their effect when the cells were from testes in mid‐spermatogenesis. In conclusion, our results suggest that in the trout extracellular adenosine and ATP are able to influence the in vitro proliferation of Go, and are potential candidates for mediating the possible influence of the nervous system on the induction, speeding up, then slowing down of spermatogenesis. Mol. Reprod. Dev. 53:443–450, 1999. © 1999 Wiley‐Liss, Inc.

Adenosine receptor-adenylate cyclase system in the trout testis: Involvement in the regulation of germ cell proliferation

“…However, this inhibition was more marked in cells from testes in mid/late spermatogenesis compared to those in early spermatogenesis (Table 2). When Ado was present in the culture medium for 4.5 days (Table 2), it had an opposite effect on cells from testes in early spermatogenesis, as it then stimulated 3 H-Tdr incorporation, as previously observed (Loir, 1999c). In the presence of 100 mM RO 20-1724, the inhibiting effect of Ado was reinforced (Table 3).…”

“…In trout, IGFs stimulate DNA synthesis by Go and primary spermatocytes (Loir and Le Gac, 1994;Le Gac et al, 1996). Recently, we found (Loir, 1999c) that the in vitro incorporation of 3 H-thymidine by trout premeiotic germ cells was stimulated by Ado present in the culture medium for 4.5 days when the cells were from testes that were starting spermatogenesis, but was inhibited when they were from testes in mid or late spermatogenesis. However, due to the well-known possible desensitization of ARs , the effects observed after such a duration of treatment with Ado may differ from short-term effects.…”

“…Because Ado, which may originate from cells within the seminiferous tubule, the circulation, and neurons, may in¯uence the spermatogenetic process (Conti et al, 1989;Loir, 1999c), the purpose of this study was to evaluate the existence of the AR±adenylate cyclase system in the trout testis and to document the potential activity and mechanism of action of Ado in the regulation of the mitotic phase of piscine spermatogenesis. This was done using primary cultures of cells from trout testes, characterized by the presence of the various somatic cell and germ cell types in a proportion which mimics that existing in situ.…”

ATP secretion in the male reproductive tract: essential role of CFTR

“…Extracellular ATP is a signalling molecule that regulates a variety of functions such as neurotransmission (Burnstock, 2007), muscle contraction (Ralevic, 2009) and water/electrolyte transport (Rieg & Vallon, 2009). In the male reproductive system, extracellular ATP is involved in several critical processes including spermatogenesis (Loir, 1999), steroidogenesis (Foresta et al 1996), sperm motility acquisition (Edwards et al 2007), epithelial ion secretion (Wong, 1988;Chan et al 1995) and smooth muscle contraction (Ruan et al 2008). In the epididymis, a small organ located downstream of the testis where sperm acquire their fertilizing abilities, extracellular ATP regulates transepithelial electrolyte and water transport (Wong, 1988;Zhou et al 2007).…”

P2X receptor channels in endocrine glands

“…ATP acts as a modulator of testicular cells, including Sertoli cells 53 and spermatogonia. 54 The Sertoli cells release ATP endogenously through a still-not clarified mechanism. 55 Leydig cells express pannexin channels, which could account for ATP release.…”