The effect of steroid hormone treatment on coho salmon (Oncorhynchus kisutch) was examined. The cDNAs for coho salmon FSH beta and LH beta subunits were cloned and sequenced using reverse transcriptase PCR. Northern blot analysis revealed that a single transcript of 1 kb for each of these subunits was present in the pituitaries of vitellogenic and spermiating coho salmon. RNase protection assays (RPAs) were developed to quantify FSH beta and LH beta subunit transcript levels. For the RPAs, antisense RNA probes and sense RNA standards were prepared from a region of the cDNAs which spanned the signal peptide and a portion of the mature protein. These RPAs were used to examine the effects of exogenous steroids including testosterone, estradiol-17beta (E2) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P) in vivo, in coho salmon at three time points during the spring period of gonadal growth when plasma levels of FSH are increasing. Both testosterone and E2 increased steady state mRNA levels of LH beta, whereas E2 decreased steady state mRNA levels of FSH beta in one experiment. Thus, the RPAs were able to detect changes in steady state mRNA levels in response to exogenous steroid treatment. Plasma and pituitary levels of FSH and LH were also measured using RIA. Throughout the experimental series, FSH plasma levels decreased in response to exogenous testosterone and E2 administration, while 17alpha,20beta-P had no effect on FSH plasma levels. Plasma LH levels were not detected throughout the course of the experiment. Pituitary LH increased in response to testosterone and E2, while pituitary FSH levels did not change. 17alpha,20beta-P had no effect on pituitary FSH or LH content during the experiment. Thus, regulation of the gonadotropins in coho salmon occurs at both the transcriptional as well as the translational level. Testosterone and E2 appear to have negative feedback effects on FSH, but positive feedback on LH.
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S J Duguay, P Swanson, and W W Dickhoff
ABSTRACT
Salmon have been shown to express alternatively spliced IGF-I mRNA transcripts coding for four different IGF-I prohormones. These transcripts, now designated Ea-1, Ea-2, Ea-3 and Ea-4, differ in size due to the inclusion of additional sequences in the E domain-coding region of the molecule. In this study, the tissue distribution and hormonal regulation of expression of alternatively spliced IGF-I mRNA transcripts were investigated in coho salmon. IGF-I mRNAs were detected by solution hybridization/RNase protection assay in all tissues examined. GH treatment significantly increased hepatic IGF-I mRNA content. Hepatic IGF-I mRNA levels were not influenced by prolactin or somatolactin. Heart, fat, brain, kidney, spleen and ovary IGF-I mRNA levels were not affected by GH, prolactin or somatolactin. Ea-1, Ea-3 and Ea-4 mRNA transcripts were detectable in the liver, and Ea-1 and Ea-3 levels increased dramatically in response to GH treatment, whereas the amount of Ea-4 mRNA was unchanged. Most non-hepatic tissues expressed only the Ea-4 transcript, and expression was not influenced by GH, prolactin or somatolactin. Ea-1 and Ea-3 transcripts were visible in gill samples from fish treated with GH. The ovaries of juvenile fish expressed Ea-1, Ea-2 and Ea-4. The amounts of these transcripts were not changed by gonadotrophin treatment. During smoltification of juvenile coho salmon, liver and gill IGF-I mRNA levels increased with increasing plasma GH and thyroxine concentrations. Muscle, brain and ovary IGF-I mRNA levels were unchanged during this period.
These data suggest that the liver is a major site of IGF-I production in response to GH. Heart, fat, brain, kidney, spleen and ovary did not show increased IGF-I mRNA levels in response to GH treatment. GH and prolactin had inconsistent effects on muscle IGF-I mRNA levels. Somatolactin and a gonadotrophin preparation did not stimulate IGF-I expression in tissues of juvenile fish. Differences in tissue GH responsiveness can be partially explained by the expression of alternatively spliced IGF-I mRNAs. Of the four hepatic IGF-I mRNA transcripts, Ea-1 and Ea-3 are GH-responsive, while Ea-2 and Ea-4 are not. Most non-hepatic tissues express only the Ea-4 transcript, and IGF-I mRNA levels do not increase after GH treatment. The increased IGF-I mRNA levels observed in gill tissue during smoltification suggest that other factors, in addition to GH, may regulate IGF-I expression. These data are also consistent with the hypothesis that IGF-I may mediate the osmoregulatory functions of GH during sea water adaptation.
