Reproductive activities of salmonids are synchronized by changes in photoperiod, which control the endocrine system via the brain-pituitary-gonadal axis. Gonadotropin-releasing hormone (GnRH) in the brain regulates synthesis and release of the pituitary gonadotropins (GTHs; FSH and LH). FSH and LH in turn stimulate the production of sex steroids for oocyte growth and maturation-Inducing steroid hormones for oocyte maturation and ovulation, respectively, in female salmonids. To clarify effects of long-term photoperiod manipulations on the reproductive activity of salmonids from early recrudescence to ovulation, we Investigated the gene expression profiles of GnRH, GTHs, and vitellogenin (VTG), and plasma sex steroids in female rainbow trout (Oncorhynchus mykiss). In addition, the percentages of eyed embryos and hatched alevins were examined together with the number of ovulated eggs to evaluate the effects of photoperiod regimes on egg quality. During late summer, the mRNA levels of GnRHs, GTHalpha, and LHbeta, and the plasma level of a maturational steroid (17alpha,20beta-dihydroxy-4-pregnen-3-one; 17,20beta-P) were significantly elevated by a gradually shortened photoperiod under constant temperature, in accordance with accelerated sexual maturation. The percentages of eyed embryos and hatched alevins from fish ovulated in August were comparable to those of control fish observed in December. These results clearly indicate that syntheses of GnRHs, LH, VTG, and 17,20beta-P are effectively accelerated by a programmed long-short photoperiod regime in early recrudescent female rainbow trout, without a marked deterioration in egg quality.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure–function relationships of G protein-coupled receptors during signal transduction.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure–function relationship of LHR–LH complexes.