Stereological studies of the effects of alpha-MSH and cAMP on melanosomes in melanoma cells.

The effects of alpha-MSH and cAMP on melanosomes in Cloudman S91 melanoma cells were investigated by modern stereological techniques. Cells were cultured for 4 days in medium containing alpha-MSH or cAMP, harvested at 24 hour intervals; some were frozen for melanin assay and the reminder embedded in Epon for light and electron microscopy. Cellular and melanosomal parameters were estimated by new stereological probes. We found that both stimulators induced increases in nuclear volume, cell volume, and the volume fractions and volumes of premelanosomes (V(Vpm,cell), Vpm) and mature melanosomes (V(Vmm,cell), Vmm), and the number of mature melanosomes (Nmm). Both stimulators also caused declines in the volume of individual mature melanosomes (V(Vimm)), the melanin content per mature melanosome unit volume and the melanin content per individual mature melanosome. The increases in the volume of individual premelanosomes and the number of premelanosomes were only induced by cAMP. The effect cAMP on some parameters occurred 24 hours prior to alpha-MSH and was more marked. The response of premelanosomes to the stimulators was more sensitive than mature melanosomes. These results suggest that both stimulators enhanced melanogenesis by increasing the V(Vpm,cell), V(Vmm,cell), Vpm, Vmm and Nmm. The melanogenic level did not depend on the V(Vimm) and melanin concentration in melanosomes. The maturation of premelanosomes was involved in melanogenesis induced by both stimulators, but, de novo synthesis and enlargement of premelanosomes were only stimulated by cAMP. It imply that exogenous cAMP may affect melanosomes, and hence melanogenesis in quantitatively or qualitatively different ways to alpha-MSH.

Application of the new stereological probes to the study of the melanosome in Cloudman S91 melanoma cells.

The relationship between melanosome size and number and melanin content has been investigated in Cloudman S91 melanoma cells growing in vitro using both "model-based" and "design-based" stereological procedures. Cells were cultured for 4 days, harvested at daily intervals, and resin-embedded for light and electron microscopy; one aliquot of each sample of cells was assayed to determine its melanin content. By comparing their volume-weighted mean nuclear volume and their number-weighted mean nuclear volume, we have found that the nuclei of Cloudman melanoma cells form a fairly homogeneous population. The volume fraction and absolute volume of premelanosomes (VVpm, cell and Vpm) and mature melanosomes (VVmm, cell and Vmm) were all found to decrease progressively throughout the period of culture as did the number of premelanosomes (Npm) and mature melanosomes (Nmm). Whilst the volume-weighted mean volume of individual stage I and stage II premelanosomes, (VVipm), remained fairly constant at about 10 nm3, the volume of individual stage III and IV mature melanosomes showed significant variation ranging between about 13 nm3 and 32 nm3. The melanin content of the cells decreased progressively over the 4 days of culture. There were, however, considerable variations in both the average melanin content per unit volume of mature melanosomes, in the range 170-600 fg/micron3, and in the melanin content per individual mature melanosome, in the range 3-12 fg. Our findings show that stereological techniques can provide unbiased and sensitive tools for the study of the morphological basis of melanogenesis; their value will become even more evident when they are combined with techniques for the localization of melanogenic enzymes and their substrates.

Preliminary report on a new method of human age estimation from single adult teeth.

A total of 317 upper and lower third molars were randomly divided into working and control samples and used to estimate chronological age employing a method which combined multiple regression analysis of data from Gustafson's and Maples's scoring system, direct morphological measurements obtained with a Kontron image analysis system and logarithmic data transformation. The standard errors of estimate were 2.4-6.8 years in the working sample and 1.9-7.5 years when the derived formulae were tested on the control sample, respectively. Compared to previous studies, this method provides a smaller standard error of estimate from a single molar tooth. The method is presently being tested on other tooth categories, like premolars, canines and other types of molars.

Stereological analysis of daily variation in the ultrastructure of the pars intermedia of the pituitary gland of the Djungarian hamster (Phodopus sungorus).

A stereological analysis has been made of the daily changes occurring in the ultrastructure of the melanotrophs of the pars intermedia of the pituitary gland of the Djungarian or Siberian hamster, Phodopus sungorus, maintained in long day photoperiods. The rough endoplasmic reticulum, Golgi apparatus and lysosomes all declined in fractional volume throughout the photophase reaching minima at mid-scotophase and rising to reach their maxima at about the time of onset of the photophase. The mitochondria reached their peak fractional volume just before the cessation of the photophase but then also declined to a minimum at mid-scotophase. No significant changes were found to occur in the fractional volumes of the nucleus or the secretory granules. These morphological findings are compared with the changes in plasma and pituitary alpha-melanocyte-stimulating hormone levels found in the rat.

Effects of alpha-melanocyte-stimulating hormone and [8-arginine]-vasotocin upon melanogenesis in hair follicle melanocytes in vitro.

alpha-Melanocyte-stimulating hormone (alpha-MSH) has been shown to act directly on the mammalian melanocyte in short-term cultures of hair follicles obtained from the Siberian hamster. Melanogenesis was stimulated through an increase in tyrosinase activity which resulted in an increase in melanin production. The response of hair follicle melanocytes to alpha-MSH occurred only in follicles taken from moulting animals, implying that they show a discontinuous expression of MSH receptors during the hair follicle growth cycle. Synthetic 1-24 ACTH had no effect on melanogenesis regardless of whether the follicles came from moulting or non-moulting animals. The pineal peptide, [8-arginine]-vasotocin (AVT), inhibited melanin production without a concomitant decrease in tyrosinase activity. In this respect AVT resembled melatonin, although AVT showed a potency ratio of less than half on a molar basis. The action of AVT, like that of melatonin, must ultimately be on some post-tyrosinase step in melanin biosynthesis. In these hair follicle melanocytes AVT seems to bind to specific receptors since neither of the closely related peptides, oxytocin and [8-arginine]-vasopressin, displayed any activity in our culture system.

Interaction of alpha-melanocyte-stimulating hormone, melatonin, cyclic AMP and cyclic GMP in the control of melanogenesis in hair follicle melanocytes in vitro.

In short-term (48 h) cultures of hair follicles alpha-melanocyte-stimulating hormone (alpha-MSH) and cyclic AMP stimulated melanogenesis through an increase in tyrosinase activity. In contrast cyclic GMP mimicked the effects of melatonin by inhibiting melanin production without causing a concomitant decrease in tyrosinase activity. Both cyclic GMP and melatonin blocked the stimulatory effects of cyclic AMP and alpha-MSH on melanin production but they left the increased levels of tyrosinase activity unaffected. Phosphodiesterase inhibitors (3-isobutyl-1--methylxanthine and papaverine) simultaneously stimulated tyrosinase activity and inhibited melanin production, presumably by allowing endogenous cyclic AMP and cyclic GMP to accumulate intracellularly. It is suggested that whereas MSH stimulates melanogenesis through a cyclic AMP-dependent mechanism there must also be an inhibitory cyclic GMP-dependent mechanism, perhaps activated by melatonin, which operates at some post-tyrosinase step in the melanin biosynthetic pathway.

Melanotrophin-potentiating factor (MPF) potentiates MSH-induced melanogenesis in hair follicle melanocytes.

Melanotrophin-potentiating factor (MPF) is a fragment of human beta-lipotrophin (LPH 88-91) which potentiates the action of alpha-MSH on Anolis skin. In the present study, we investigated the effect of MPF on MSH-induced melanogenesis. Pooled hair follicle scrapings from Siberian hamsters (Phodopus sungorus) were incubated for 48 hours with or without alpha-MSH and/or MPF. Melanogenesis was monitored by measuring tyrosinase activity and melanin accumulation. 10-8 M MPF potentiated the effect of no effect on melanogenesis, but 10-9 to 10-7 M alpha-MSH caused a dose-related increase. 10-8 M MPF potentiated the effect of each dose of alpha-MSH. Thus MPF potentiated MSH action on mammalian melanogenesis as well as on reptilian melanosome dispersion. Although each of these processes involve different intracellular responses the receptor mechanisms involved in each may therefore be the same.

Photoperiodic dependence of seasonal changes in pituitary content of melanocyte-stimulating hormone.

In the Siberian hamster the pituitary content of bioassayable MSH has been shown to be seasonally variable, being greater during the long days of spring and summer than during the short days of autumn and winter. Similar changes in the pituitary content of both bioassayable MSH and immunoreactive alpha MSH can be induced by transfer of animals between artificial light photoperiods of appropriate duration.

Post-tyrosinase inhibition of melanogenesis by melatonin in hair follicles in vitro.

In short-term (48 hr) culture hair follicles of the Siberian hamster retain both tyrosinase activity and the capacity to produce melanin. The addition of melatonin to such cultures at concentrations between 10-6 M and 10-10 M brings about a dose-related inhibition of melanogenesis but tyrosinase activity is unaffected. The use of a series of melatonin analogues and blockers suggests that the hair follicle melanocytes possess melatonin receptors, although their location remains to be determined. Melatonin also inhibits the increase in melanogenesis brought about by alpha-melanocyte-stimulating hormone (MSH) but again it has no effect upon the increased levels of tyrosinase which accompany this MSH response. It is suggested that melatonin inhibits melanogenesis through a mechanism which operates at some post-tyrosinase step in the melanin biosynthetic pathway.

Pelage color cycles and hair follicle tyrosinase activity in the Siberian hamster.

Hair follicle tyrosinase levels and melanin content, together with serum tyrosine levels have been studied in relation to annual pelage color changes in the Siberian hamster. Tyrosinase levels were found to peak not only at the spring moult when pigmented hair is produced but also at the autumn moult when the hair produced is unpigmented. The melanin content of hair follicles was high in summer and low in winter but serum tyrosine levels did not differ at the spring and autumn moults. These results suggest that some factor must exist to prevent the raised tyrosinase levels of the autumn moult being expressed as melanogenesis and that this factor must be photoperiodically modifiable, being controlled through a neuroendocrine mechanism.

Early changes in the ultrastructure of the pars intermedia of the pituitary of Xenopus laevis after change of background color.

Stereological analysis of the secretory cells of the pars intermedia of Xenopus laevis over a period of 3 days following the transfer of animals from a white to a black background has revealed that significant alterations in the ultrastructural appearance of these cells can be detected 8 h after the transfer. In particular, changes in the secretory granules and the rough endoplasmic reticulum were found to correlate well with previous reports concerning the melanocyte-stimulating hormone (MSH) content and the capacity for protein synthesis of the pars intermedia.