On the one hand levels
On the one hand, levels of up to 20 nM P4 have been reported for a cyprinid species, Labeo rohita, in summer (Suresh et al., 2008). Similarly, P4 concentrations in tilapia, Oreochromis mossambicus, were found to range up to 27 nM (Cornish, 1998). Nevertheless in sturgeon P4 levels of approximately 3 nM have been reported (Barannikova et al., 2002). On the other hand, DHP4 was found to be as high as 15 nM in the cyprinid, Labeo rohita (Sen et al., 2002). Moreover, induction of spawning by using injection of carp pituitary extract increased circulating DHP4 levels up to 300 nM in carp (Yaron and Levavi-Zermonsky, 1984). Furthermore, in stimulated mammalian leukocytes P4 was reported to inhibit iNOS expression and NO production in vitro and in vivo (Miller et al., 1996, Coughlan et al., 2005) and similar effects have been reported for carp in vitro and in vivo (Yamaguchi et al., 2001, Watanuki et al., 2002) but steroid mode of action was not investigated. In our study, especially the natural progesterones P4 and DHP4, used at concentrations which may partly be considered to be environmentally relevant, impaired NO production of head kidney leukocytes. This was also obvious for the synthetic progestin MPA, and trunk kidney leukocytes were sensitive to this substance as well. Moreover, the high sensitivity of trunk kidney-derived immune Merimepodib and the higher NO levels produced by these cells compared to head kidney-derived leukocytes suggest that the trunk kidney-derived leukocytes constitute of more mature immune cells than head kidney preparations. However, the synthetic progestin LEV did not significantly change NO production of head and trunk kidney leukocytes. These findings are not surprising for LEV is a 19-nortestosterone derivative that has already been reported to exert contrasting effects to typical progesterone and pregnane derivatives used in contraception (van der Vange et al., 1990). This may be due to the fact that LEV is thought to have no binding affinity to membrane-associated receptors for progestogens (Luconi et al., 1998). Our goal was to use a powerful combination of biochemical and molecular approaches to define the mechanisms and functions of progestogens in immune regulation in fish. In general, steroid hormones regulate target genes at the level of transcription. To investigate regulation of gene expression by progestogens we also determined mRNA expression of iNOS. Though, systematic evaluation of reference genes is lacking in fish, we normalised target gene mRNA expression to b2m which was reported to be a suitable reference gene in gene expression studies in human immune cells (Zhang et al., 2005). Congruently, b2m mRNA expression seems to be suitable as a reference gene because it did not differ between head and trunk kidney samples (Wilcoxon test, p=0.753) and was not significantly influenced by steroid treatment (Friedmann test, p=0.647 in head kidneys and p=0.769 in trunk kidney leukocytes; data not shown). Constant expression in different tissues is recommended for reference genes. To our knowledge, this is the first study using b2m for normalisation in studies in fish immunology. The iNOS mRNA expression was reduced by some progestogens. However, a significant difference between unstimulated and stimulated cells after 96 h of incubation was only observed in head kidney-derived leukocytes. This is probably due to the fact that the time point of expression analysis can be considered as being too late for induction of iNOS expression which was reported to be already decreased after 24 h of stimulation in fish (Laing et al., 1999). Thus, more pronounced effects of steroids on iNOS expression would be expected at an earlier time point of measurement. Whether there is also an effect of steroids on iNOS protein content in carp leukocytes as shown for mammals (Coughlan et al., 2005) remains to be investigated. Since it has been concluded that P4 exerts its effects on cytokine production and macrophage activation through inhibition of NFκB in mammals (Miller and Hunt, 1996) an influence of progestogens on cytokine production in carp leukocytes which may result in the effects on iNOS expression can be considered. Nevertheless, further investigations on effects of progestogens on leukocyte functions are needed to fully understand the impact of these steroidal compounds on immune cell activity and other physiological processes to elucidate these essential characteristics of the immune defense in fish. Moreover, it should be noted that immunosuppressive effects of progesterones e.g. in the spawning season should be considered to be in the animal's interest since they regulate distribution of available resources among tissues with competing demands. This allows animals to save energy to attract mates and for successful reproduction (Ottová et al., 2005). However, also neuroprotective actions of P4 have been proposed in mammals (Labombarda et al., 2003).