Immunocytochemical localization of melatonin in the harderian gland of Syrian hamster.

BACKGROUND: The Harderian gland (HG) is a tubulo-alveolar gland found within the ocular orbit of animals which present a nictitating membrane. The Harderian gland is regarded as an extrapineal melatonin producing organ and both, photoperiod and melatonin have been shown to exert an important role in the metabolism and morphological features of such a gland. Our results seem to support the presence of melatonin in the nuclei of the HG cells, although our studies have not definitively proved such presence. METHODS: An Immunocytochemical anti-melatonin technique was done over free sections of Bouin fixed material obtained from Syrian hamsters. Some of the sections were embedded in an epoxy resin and studied under electron microscope. RESULTS: The presence of positive immunoreaction was observed at the level of the nuclear membranes and in close relation to chromatin. No differences were observed between males and females nor between pinealectomized animals and control ones. CONCLUSIONS: Our results suggest the binding of melatonin to the cell nucleus in all cell types of the gland. These observations are in accord with the binding studies performed by Acuña-Castroviejo in purified cell nuclei of rat liver (Acuña-Castroviejo et al., 1994. J. Pineal Res., 16:100-112) and the earlier one by Menéndez-Peláez et al. (1993a,b, J. Pineal Res., 15:59-69; J. Cell Biochem., 53:373-3*2) using the light microscope. Our results seem to support the idea of a nuclear action of melatonin and they agree with the observations of Carlberg and Wiesenberg (1995, J. Pineal Res., 18:171-178) about the activation of some orphan receptors by melatonin.

Photoperiod and the pineal gland regulate the male phenotype of the Harderian glands of male Syrian hamsters after androgen withdrawal.

The Harderian glands of Syrian hamsters exhibit a marked sexual dimorphism in cell types and porphyrin production. The glands of male hamsters have two secretory cell types (Type I and II) while the glands of females consist of a single secretory cell type (female Type I) and large intraluminal deposits of porphyrins. Besides androgens, there is evidence that the pineal gland, through the secretion of melatonin, contributes to the maintenance of the "male" and "female" phenotypes. In this study, we investigated the effects of castration, short photoperiods, and pinealectomy on the distribution of secretory cells and porphyrin deposits in the Harderian glands of male Syrian hamsters. Two groups of animals were maintained in long days (14 hr light/day). Hamsters in one group were left intact and those in the other were castrated. Another three groups were maintained in short days (8 hr light/day); these animals were either left intact, castrated, or both castrated and pinealectomized. The duration of the experiment was 5 weeks. Castration of long photoperiod-exposed animals resulted in a significant drop in the number of Type II cells and a large increase in the porphyrin deposits (P < 0.01). However, castrated animals exposed to short photoperiod showed a significant smaller change in both parameters compared with those exposed to long days (P < 0.05). Pinealectomy prevented the effects of short days in castrated animals. No significant changes were observed in the relative number of mitotic figures or in the number of cell nuclei, indicating that the changes observed were due in part to a transformation of Type II into Type I cells. In a second experiment, male hamsters were injected daily either with 25 micrograms of melatonin late in the afternoon or with the saline for 8 weeks. The administration of melatonin resulted in a significant (P < 0.05) increase in the percentage of Type II cells. We conclude that when circulating androgens are very low or absent, pineal melatonin maintains the male phenotype in the Syrian hamster Harderian gland.

Chronic N-methyl-D-aspartate administration prevents melatonin-associated changes in cell differentiation in the harderian glands of male hamsters.

The daily administration of 25 micrograms of melatonin for 10 weeks resulted in an increase in the percentage of Type II cells in the Harderian glands of male Syrian hamsters. Harderian glands of melatonin injected animals consisted of 65-70% Type II cells while control animals which were injected with saline had 40% Type II secretory cells. The daily administration of 3 mg of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) prevented the effects of melatonin on cell differentiation but was without effect when administered to saline treated hamsters alone. Both the relative number of mitoses and the number of total cells, estimated by counting the nuclei, was not affected. Thus, a conversion from Type I to Type II cells seems possible. The effects of melatonin and NMDA administration were independent of the serum levels of testosterone, luteinizing hormone and thyroxine, hormones which have been implicated in Type II cell differentiation. However, prolactin levels, which were affected by melatonin and NMDA administration, might be involved in the differentiation of Harderian gland secretory cells.