Basic fibroblast growth factor (bFGF) plays an important role in development of the central nervous system and is neurotropic for a variety of neurons. In this study, we investigated whether bFGF is neurotropic for GT1 GnRH neuronal cell lines and if these cells express functional FGF receptors (FGFRs). The GT1 cell lines generated by genetically targeted tumorigenesis display highly differentiated properties of GnRH neurons. Addition of 2 and 10 ng/ml bFGF increased neurite outgrowth of GT1-7 cells and resulted in a significant increase of GT1 cell survival in serum-free medium. However, bFGF had no effect on [3H]thymidine incorporation at 24 or 48 h. RNase protection assays using riboprobes specific for murine FGFRs 1-3 showed that GT1 cells express FGFRs 1 and 3 but not 2. Occupancy of FGFRs with 10 ng/ml bFGF stimulated the sustained tyrosine phosphorylation of both the 42- and 44-kilodalton mitogen-activated protein kinases (MAPKs) for up to 6 h as shown by Western blot analysis. In addition, phosphorylation of the MAPKs was associated with enzyme activation as shown by an in-gel MAPK assay. GT1-1 and GT1-7 cells also express messenger RNA for bFGF, although the level of bioactive bFGF synthesized by GT1 cells appears suboptimal because GT1 cells can further respond to exogenously added bFGF. Thus, we have demonstrated that bFGF is a neurotropic factor in GT1 GnRh neuronal cell lines, raising the possibility that bFGF may play a role in the neurobiology of GnRH neurons.

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2,-4.3, and-5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.

Both 17 beta-estradiol (E2) and testosterone (T) were shown to inhibit in vitro pituitary LH secretion in the turtle Trachemys scripta. Since T was approximately 500 times less potent than E2, and 5 alpha-dihydrotestosterone was even less active than T, the inhibitory action of T may result from its aromatization to estrogen. We utilized both in vivo and in vitro approaches to elucidate the roles of T and estrogen in the negative feedback of pituitary LH secretion. Gonadectomy of adult (vitellogenic) females significantly elevated plasma LH. Adult females treated with fadrozole (an aromatase inhibitor) with or without daily injections of keoxifene (an antiestrogen) also showed an increase in plasma LH to a level comparable to that observed in gonadectomized females, whereas plasma LH levels of juvenile females treated with fadrozole remained undetectable. In vitro LH secretion in response to GnRH in juvenile females was significantly inhibited by 48-h exposure to 50 ng/ml T or 100 pg/ml E2. Both fadrozole (200 microM) and keoxifene (200 nM) significantly blocked this T-induced inhibition of LH secretion, demonstrating that T lacks intrinsic inhibitory activity. Confirmation of the inhibition of aromatase activity by fadrozole comes from metabolic studies of 1 beta-[3H]androstenedione using turtle brain, ovary, and pituitary. In vitro, fadrozole altered the metabolism of 1 beta-[3H]androstenedione and inhibited aromatase activities in these tissues. These results indicate that the inhibitory effect of T is largely mediated through its aromatization to estrogen, and that estrogen is primarily responsible for the suppressed LH secretion in vitellogenic adult turtles.