Regulation of the connexin 43 promoter in the brook trout testis: role of the thyroid hormones and cAMP.

Gap junctions are critical for spermatogenesis. They are composed of integral proteins, the connexins. In mammals, a loss of Cx43 expression results in the inhibition of spermatogenesis. We have shown that Cx43 is expressed in the Sertoli cells of rainbow trout and that cAMP and triiodothyronine (T(3)) regulate testicular Cx43 expression in brook trout testis. The objective of this study was to determine if cAMP and T(3) act at the level of the cx43 promoter to regulate its expression. A 607 bp 5' flanking sequence of the cx43 promoter was obtained by Genome Walking. A TATA box was predicted to be located between positions -36 and -30 relative to the transcriptional initiation site. 5'-Rapid amplification of cDNA ends indicated a single transcriptional start site. Single C/EBP (-164 to -156) and tr-beta (-112 to -107) response elements were identified and electrophoretic mobility shift assays indicated the presence of competitive protein binding sites at each region. Immortalized rainbow trout gonadal cell line (RTG-2) which express cx43 and tr-beta transcripts were transfected with a vector containing the Cx43 promoter inserted into a luciferase expression vector. Transactivation of the reporter genes was stimulated by either cAMP or T(3). Sequential deletion and point mutations in either the C/EBP or tr-beta response element indicated that T(3) but not cAMP directly induced luciferase transactivation of the luciferase gene by acting on different sites of the Cx43 promoter. Together, these data indicate that T(3) stimulates cx43 expression via direct regulation of gene transcription.

Seasonal variations in testicular connexin levels and their regulation in the brook trout, Salvelinus fontinalis.

Spermatogenesis requires coordinated intercellular communication mediated by gap junctions. Gap junctions are composed of connexons that are themselves composed of connexins (Cxs). The present objective was to determine the regulation of testicular Cxs in a seasonal breeder, the brook trout. To assess seasonal variations in testicular Cxs, trout were sampled monthly throughout spermatogenesis (June-November). Circulating levels of testosterone (T) and 11-ketotestosterone (11-KT) as well as mRNA levels for testicular androgen receptors (ar-alpha, ar-beta), thyroid hormone receptors (tr-alpha, tr-beta) and gonadotropin I receptor (rgthI) were measured. Plasma T levels peaked in October, one month prior to spawning, while 11-KT levels peaked at spawning. ar-alpha and ar-beta mRNA levels increased during spermatogenesis and peaked in November while tr-alpha, tr-beta mRNA levels stayed constant throughout spermatogenesis and increased dramatically in November. rgthI mRNA levels decreased progressively during spermatogenesis. Cx43 and Cx30 levels were constant during spermatogenesis and decreased in November. Cx31 levels were also constant during spermatogenesis but decreased dramatically in October and November. Cx43.4 levels peaked in July then decreased in September and levels were undetectable thereafter. Using in vitro cultures of testicular fragments we demonstrated that cx43 mRNA levels were regulated in a dose-response manner by 3,5,3'-triiodo-l-thyronine (0-370 nM) and cAMP (0-100 ng/ml) but levels were not regulated by 11-KT. These results indicate that testicular Cxs vary as a function of spermatogenesis and that the expression of cx43 in the trout testis is regulated by both cAMP and TH.

Influence of municipal effluents on the expression of connexins in the brook trout (Salvelinus fontinalis) testis.

Gap junctions are essential for spermatogenesis. Exposure to municipal wastewater effluent can modify spermatogenesis in fish. The present aim was to determine if municipal wastewater effluent could alter the expression of testicular connexins (Cxs) in brook trout. Trout were exposed for 4 and 12 weeks to various concentrations of municipal effluent (0, 1, 10 and 20%, v/v). Hepatic vitellogenin (vtg) mRNA levels were not different between exposure groups after 4 weeks. At this time, testicular cx43 and cx31 mRNA levels increased in the 1% group, but cx30 and cx43.4 levels were not different at any concentration. Immunolocalization of each Cx did not differ between groups after 4 weeks. After 12 weeks, spermatogenesis in the 1% group was more advanced than in other groups, and hepatic vtg mRNA levels were significantly increased at the higher exposure concentrations. Testicular cx43 mRNA levels were higher than controls at all doses, while cx43.4 levels increased in a dose-dependent manner but remained lower than controls. cx31 mRNA levels were significantly lower in the 1 and 10% groups than in control and 20% group, while cx30 levels did not vary. Immunolocalization of Cxs did not differ between groups except for Cx43.4, which was expressed between spermatocytes in the 1% group. Furthermore, the Cx31 immunoreaction appeared to decrease in testicular blood vessels of fish exposed to the highest dose. Furthermore, vegf mRNA levels were unaltered by treatment at both time points. Thus, long-term exposure to environmental concentrations of wastewater effluent can alter the expression of testicular Cxs.

The expression of multiple connexins throughout spermatogenesis in the rainbow trout testis suggests a role for complex intercellular communication.

Certain fish, such as rainbow trout (Oncorhynchus mykiss), are seasonal breeders. Spermatogenesis in rainbow trout is synchronous; therefore, at any time point during this process, germ cells are predominantly at the same stage of development. As such, rainbow trout represent an excellent model in which to study spermatogenesis. Gap junctions are composed of connexons, which are themselves formed by six transmembrane proteins termed connexins (Cxs). The objectives of this study were to assess which Cxs are expressed in the rainbow trout testis, and if their expression was stage specific during gonadal maturation. Rainbow trout were killed at various stages of maturation, and total cellular RNA was isolated from the testes. RT-PCR using degenerate primers recognizing all vertebrate Cxs indicates that there are several different Cxs in trout testes. Amplicons were cloned and sequenced. Homology comparisons indicate that these were cx43, cx43.4, cx31, and cx30. Immunolocalization of these Cxs indicate that Cx43 was localized primarily to Sertoli cells, while Cx43.4 was localized along the lateral plasma membranes between adjacent spermatocytes. Cx30 was localized to the interstitial Leydig cells, and Cx31 was localized primarily to the endothelium of interstitial blood vessels. The expression of each Cx varied as a function of the stage of spermatogenesis, suggesting that the expression of these proteins is highly regulated. Together, these results indicate that intercellular communication in the testis is complex, involves several different Cxs, and is a highly regulated process.