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Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.

Estrogen exerts a variety of important physiological effects, which have been suggested to be mediated via the two known estrogen receptors (ERs), alpha and beta. Three-month-old ovariectomized mice, lacking one or both of the two estrogen receptors, were given estrogen subcutaneously (2.3 micro g/mouse per day) and the effects on different estrogen-responsive parameters, including skeletal effects, were studied. We found that estrogen increased the cortical bone dimensions in both wild-type (WT) and double ER knockout (DERKO) mice. DNA microarray analysis was performed to characterize this effect on cortical bone and it identified four genes that were regulated by estrogen in both WT and DERKO mice. The effect of estrogen on cortical bone in DERKO mice might either be due to remaining ERalpha activity or represent an ERalpha/ERbeta-independent effect. Other effects of estrogen, such as increased trabecular bone mineral density, thymic atrophy, fat reduction and increased uterine weight, were mainly ERalpha mediated.

Estrogen treatment has positive effects on the skeleton, and we have shown that estrogen receptor alpha (ERα) expression in cells of hematopoietic origin contributes to a normal estrogen treatment response in bone tissue. T lymphocytes are implicated in the estrogenic regulation of bone mass, but it is not known whether T lymphocytes are direct estrogen target cells. Therefore, the aim of this study was to determine the importance of ERα expression in T lymphocytes for the estrogenic regulation of the skeleton using female mice lacking ERα expression specifically in T lymphocytes (Lck-ERα^−/−) and ERα^flox/flox littermate (control) mice. Deletion of ERα expression in T lymphocytes did not affect bone mineral density (BMD) in sham-operated Lck-ERα^−/− compared to control mice, and ovariectomy (ovx) resulted in a similar decrease in BMD in control and Lck-ERα^−/− mice compared to sham-operated mice. Furthermore, estrogen treatment of ovx Lck-ERα^−/− led to an increased BMD that was indistinguishable from the increase seen after estrogen treatment of ovx control mice. Detailed analysis of both the appendicular (femur) and axial (vertebrae) skeleton showed that both trabecular and cortical bone parameters responded to a similar extent regardless of the presence of ERα in T lymphocytes. In conclusion, ERα expression in T lymphocytes is dispensable for normal estrogenic regulation of bone mass in female mice.

Estrogens are important for the male skeleton. Osteoprotegerin (OPG), receptor activator of NF-kappa B ligand (RANKL), interleukin-6 (IL-6), IL-1 and tumor necrosis factor alpha (TNFalpha) have been suggested to be involved in the skeletal effects of estrogen. We treated orchidectomized mice with estradiol for 2 weeks and observed a 143% increase in the trabecular bone mineral density of the distal metaphysis of femur that was associated with a decreased OPG/RANKL mRNA ratio in vertebral bone. A similar decreased OPG/RANKL ratio was also seen after estrogen treatment of ovariectomized female mice. The effect of estrogen receptor (ER) inactivation on the OPG/RANKL ratio was dissected by using intact male mice lacking ER alpha (ERKO), ER beta (BERKO) or both receptors (DERKO). The expression of OPG was increased in ERKO and DERKO but not in BERKO male mice, resulting in an increased OPG/RANKL ratio. Furthermore, serum levels of IL-6 and tartrate-resistant acid phosphatase 5b (TRAP 5b) were decreased in ERKO and DERKO, but not in BERKO male mice. These results demonstrate that ER alpha, but not ER beta, is involved in the regulation of the vertebral OPG/RANKL ratio, serum levels of IL-6 and TRAP 5b in male mice.

Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17β-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.