Tyrosinase activity (TH, DO, PAGE-defined isozymes) and melanin production in regenerating hairbulb melanocytes of lethal yellow (Ay/a), black (a/a), agouti (AwJ/AwJ/) and albino (a/a/c2J/c2J) mice (C57BL/6J)

We compared tyrosinase activity (TH, DO, and native PAGE-defined isozymes) and melanin production in particulate and soluble fractions of hairbulb melanocytes of lethal yellow (Ay/a C/C), nonagouti black (a/a C/C), and albino (a/a c2J/c2J) of 3-, 6-, 9-, and 12-day regenerating hairbulbs. With respect to tyrosine hydroxylase (TH) and dopa oxidase (DO) activities, Ay/a melanocytes possessed only 25-35% of the activity of a/a; there were no genotype differences in either the subcellular distribution of activity in soluble and particulate fractions or in the relative increases of activity over the 12-day developmental period. TH data on wild-type agouti (AwJ/AwJ) mice over the 3-11 day regeneration interval showed an activity intermediate between that of a/a and Ay/a; the rate of TH increase reflected black and yellow phases of the agouti hair cycle. Analyses of the number and densities of dopa-sensitive bands following native PAGE of 3-, 6-, 9-, and 12-day hairbulb fractions of a/a and Ay/a mice suggested stage-dependent patterns. A comparison of rates and amounts of melanin production in 3-, 6-, 9-, and 12-day fractions showed consistent melanin production in Ay/a to be 10-20% that of a/a; however, fold increases in melanin production over the four stages were similar between genotypes. Overall, tyrosinase activity data support the notion that agouti locus modification of tyrosinase activity is a graded or quantitative rather than a qualitative phenomenon.     

Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.     

Function of the hypothalamo-pituitary-adrenal system in mice with ectopic overexpression of agouti protein

Agouti protein (AP) is known to antagonise the effects of melanocortins (ACTH, MSH) on the melanocortin receptors which participate in regulation of central and peripheral HPA links. This study aimed at estimation of effects of dominant mutation Agouti yellow (Ay-ectopic overexpression Agouti protein) on the HPA axis function in mice. Male mice of C57B1/6J strain of Ay/a- and a/a genotypes (control animals, lack of AP), were used. We demonstrated that basal corticosterone level in the Ay mice matched those of control animals. Stress-activated corticosterone level (p < 0.02) and sensitivity of adrenal to low doses ACTH in vitro and in vivo were higher in Ay mice compared with control mice. Dexamethazone-inhibition of stress-reactivity was more intensive in Ay/a than in a/a mice (p < 0.0007).     

Effects of dehydroepiandrosterone on obesity and glucose-6-phosphate dehydrogenase activity in the lethal yellow mouse (strain 129/Sv-Ay/Aw)

We investigated the anti-obesity effects of the adrenal androgen, dehydroepiandrosterone (DHEA), on genetically predisposed obese lethal yellow mice (Ay/Aw). Secondly, we tested the hypothesis that DHEA promotes its anti-obesity effects by decreasing the activity of glucose-6-phosphate dehydrogenase (G6PDH). We subjected four genotype-sex combinations of yellow and agouti (control) mice to four dietary treatments and determined weight changes, food consumption, and G6PDH activity. Although G6PDH activities of yellow mice were considerably decreased in the 0.4% DHEA treatment group, they were elevated in the 0.0 and 0.1% DHEA treatment groups. In contrast, G6PDH activities of DHEA-treated control agouti mice remained relatively constant. These studies confirm that DHEA prevents the Ay gene from promoting excess fat deposition via some mechanism(s) other than reduced dietary intake. However, the overall absence of agreement between weight change (gain or loss) and G6PDH activity suggests that the anti-obesity activity of DHEA is not mediated via G6PDH. Since yellow obese (Ay/Aw) mice were found to be more susceptible to DHEA's effects than their agouti (Aw/Aw) littermates, Ay appears to induce an altered metabolism in Ay/Aw mice which is more susceptible to the effects of DHEA than the normal metabolism of Aw/Aw mice.     

Effects of reciprocal ovary transplantation on reproductive performance of lethal yellow mice (Ay/a; C57BL/6J)

This study was conducted to determine whether reproductive failures in ageing, obese lethal yellow (Ay/a) females are due primarily to defects within Ay/a ovaries or to systemic defects which may operate outside the ovaries. Reciprocal ovary transplantation between control (a/a) and lethal yellow (Ay/a) females provided an experimental system to test the reproductive potential of not only Ay/a ovaries in control (a/a) females but also control (a/a) ovaries in mutant (Ay/a) females. Results on reproductive performance of all four combinations of grafts between Ay/a and a/a mice proved that Ay-induced reproductive failures are not due to intrinsic ovarian lesions but rather to defects operating extrinsically to the ovary. The hypothalamo-pituitary axis is a likely site for this reproductive lesion.     

Effects of diapause on lethal yellow (Ay/Ay) mouse embryos.

One of the problems in studying early acting recessive lethal genes is recognizing the homozygotes prior to their demise. Molecular probes can assist in this task, but their use generally requires removal of cells and consequent damage to the embryo, which may compromise its subsequent development. The present study was undertaken in an attempt to distinguish intact, living, lethal yellow (Ay/Ay) embryos from Ay/ae and ae/ae littermates before implantation by placing them in implantation delay or diapause. After 2 days in the reproductive tract of prepubertal females, the great majority of presumptive lethal Ay/Ay embryos has failed to hatch from the zona pellucida and they exhibited a marked deficiency of cells relative to controls, particularly in the inner cell mass. This argues against a stage-specific role for the gene in implantation.     

The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis

The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis.     

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.     

Differentiation of hairbulb pigment cell melanosomes in compound agouti and albino locus mouse mutants (Ay, a, c2J; C57BL/6J)

Our objective was to determine using electron microscopy how nonagouti (a), lethal yellow (Ay), and albino (c2J) genes affect the program of mouse hairbulb melanosome differentiation; 1,921 hairbulb melanosomes from four genotypes (a/a C/C = B,Ay/a C/C = Y, a/a c2J/c2J = BA, and Ay/a c2J/c2J = YA) were scored for developmental stage, length, and width. Qualitative and quantitative electron microscopy revealed the following. An albino locus-induced diminution of melanosome size suggests that the albino locus is involved in structural features of melanosomes not directly related to the synthesis and deployment of tyrosinase. Ratio data on melanosome length-to-width confirm that the agouti locus determines melanosome shape, either spherical or elliptical; melanization is not required for melanosomes to achieve their agouti-locus-determined shapes. YA (Ay/a c2J/c2J) melanosomes, characterized by poorly organized matrices, absence of active tyrosinase, unusually large membrane invaginations, and significantly smaller dimensions than those of BA (a/a c2J/c2J), showed additive effects of both Ay and c2J alleles. These data suggest that the albino locus plays a structural as well as functional (tyrosinase) role in the differentiation of mouse hairbulb melanosomes. The agouti locus, even in the absence of melanization, directs melanosome shape either via synthesis and deployment of agouti-locus-encoded matrix proteins or by other structural factors. The additive effects of Ay and c2J alleles in compound YA mutants document the importance of specific interactions both functional and structural between agouti and albino loci.     

Investigation of the tissue specificity of the lethal yellow (Ay) gene in mouse embryos

The tissue specificity of the lethal yellow mutant was investigated by separation of blastocyst tissues. Embryos from experimental (Ay/ae X Ay/ae) and control (ae/ae X Ay/ae) crosses of the AG/CamPa inbred strain were recovered at 3.5 days post coitum, cultured for 24 hours, and then mechanically dissected into the component tissues of the blastocyst, the inner cell mass (ICM), and trophectoderm. These fragments were then cultured separately, with or without a feeder layer of inactivated fibroblasts, for an additional 3-5 days. Comparisons between experimental and control crosses indicated that the lethal Ay/Ay embryos were among the blastocysts successfully dissected but that both the ICM and trophectoderm from lethal embryos failed to develop further in vitro, either with or without feeders. With retrospective identification of the lethal embryos, it was found that at 4.5 days, after 1 day of culture, they had formed morphologically normal blastocysts but were frequently more fragile upon dissection and had smaller ICMs. Although none had hatched from the zona pellucida, some had ruptured it and were halfway out. With culture, lethal ICMs showed no development, and lethal trophectoderm usually attached but showed very limited outgrowth. Thus, no rescue of lethal tissue was shown with dissection and in vitro culture, and results are consistent with the gene affecting both tissues of the late blastocyst.     

Overproduction of agouti-protein did not affect cAMP level but increased steroidogenesis in adrenal glands under forskolin stimulation

Mutation Agouti yellow (Ay) in mice Ay/a results in overproduction of agouti protein (AP), adult onset of obesity, increased corticosterone responses to restrain stress as compared with a/a mice (absence of AP). The enhanced corticosterone response in restrained Ay/a-mice compared with restrained a/a-mice occurred in result of increased adrenal reactivity to ACTH. The purpose of this work was to investigate the influence of AP overproduction on adenylate cyclase (AC) activity and steroidogenesis in forskolin stimulated adrenal cells. To estimate obesity influence, these parameters were measured in young (3 weeks) and adult (15 weeks) animals. The data obtained demonstrated that AP overproduction and the obesity did not affect the AC activity. However, forskolin stimulated corticosterone production in Ay/a-mice was higher than in a/a-mice (in young--during 0.5 h, in adult--during 3 hrs of incubation). So AP overproduction and obesity affect the corticosterone production. We hypothesize that AP overproduction affects steroidogenesis gene expression: accelerates gene activation in ontogenesis and increases enzyme de novo synthesis during long-term stimulation in adults.     

Steroidogenesis in adrenal glands of young and adult female mice with hyperexpression of the Aguoti gene

Dominant mutation Agouti yellow (AY) leads to ectopic overexpression of the Agouti gene and yellow coat color in mice. Furthermore, the mutation Ay increased adrenal response to emotional stress. The study assessed whether pleiotropic effect of the mutation Ay on adrenals function was dependent on sex and age. 3- and 15-week old female C57B1/6J mice of two agouti-genotypes: Ay/a (ectopic Agouti-gene overexpression) and a/a (absence of Agouti-protein), were investigated. Cyclic AMP level (adenylate cyclase activity) and corticosterone production in adrenal isolated cells stimulated by ACTH and dibutyrul cAMP (db-cAMP) were measured. ACTH increased cAMP accumulation to the same extent in Ay/a- and a/a-mouse adrenal cells of both ages. The dibutyrul cAMP-induced corticosterone production was higher in Ay/a than in a/a-mouse adrenal cells of both ages. The ACTH-induced corticosterone production in 3-week- old Ay/a-m/CQ was lower and in 15-week old Ay/a-mice was higher than in a/a-mice of the respective ages. The ACTH- and db-cAMP-induced steroidogenesis was not changed in Ay/a-mice and decreased in a/a-mice with age. Thus, in females as well as in males, the mutation Agouti yellow did not affect adenylate cyclase activity, increased db-cAMP-induced corticosterone production and disturbed development of adrenal cortex.     

In vitro analysis of pre- and early postimplantation development of lethal yellow (Ay/Ay) mouse embryos

Preimplantation embryos from matings between yellow heterozygous (Ay/a) mice were recovered at 56 hours post coitum, cultured for five days, and compared with the development of embryos from three control matings (Ay/a female X a/a male, a/a female X Ay/a male, a/a female X a/a male). Most embryos were at the 8-cell stage at recovery; however fewer embryos from the experimental cross had developed to the 8-cell stage than embryos of control matings, indicating a developmental lag of experimental embryos (P less than 0.01). The yellow (Ay/a) uterus did not contribute (P = 0.05) to delayed development. Experimental and control embryos were equally capable of successful development in culture to the morula stage with no distinct morphological characteristics identifying the class of Ay/Ay mutants. However, significant differences were observed in the development from morulae to blastocysts; 9.4% (10/106) of the morulae in experimental crosses failed to undergo blastocyst formation as compared with 2.5% (10/398) of morulae in pooled control crosses (P = 0.010-0.025). In the experimental cross 25.0% (24/96) of embryos that developed successfully to the blastocyst stage failed to hatch from the zona pellucida; these are presumed to include the class of lethal yellow homozygotes. Abnormalities seen in cultured embryos consisted primarily of blastomere disintegration, blastomere arrest and exclusion, and embryo fragmentation.     

Genetic controls over melanocyte differentiation: interaction of agouti-locus and albino-locus genetic defects

Tyrosinase activities and dopachrome conversion activity were evaluated in extracts made from skins of 6-day-old mice that were mutant at the agouti and albino loci. Dopa oxidase (DO) activity of tyrosinase in fully pigmented (C/C) mice is reduced in extracts made from skins of yellow 6-day-old mice as compared to those of black mice. Dopachrome conversion (DC) activity is absent from skin extracts of normal yellow mice and is present in normal black mice. DC activity is a characteristic of a separate enzyme which has been called dopachrome conversion factor or dopachrome oxidoreductase. We measured the dopa oxidase activity and dopachrome conversion activity in skin extracts of yellow mice and black mice that were mutant at the albino (C) locus. Extracts made from extreme-dilution (ce/ce) mice do not have DO activity. Those from yellow extreme-dilution mice do not have DC activity, while those from black, extreme-dilution mice do. The DO and DC activities that characterize skin extracts made from platinum (cp/cp) yellow mice are similar to those of platinum black mice. These observations suggest possible mechanisms by which the functions controlled by the agouti and albino loci interact to control melanogenesis.     

Decay of hormone responsiveness in mouse melanoma cells in culture as a function of cell density

Cloudman S91 mouse melanoma cells lose their ability to demonstrate an MSH-induced increase in tyrosinase activity as cell density increases. This loss in hormone responsiveness occurs before confluency is reached and cannot be reversed by exposure of cells to increasing concentrations of MSH. The failure of high-density cultures to respond to MSH is apparently not the result of an inability of MSH to stimulate cAMP production, since either low- or high-density cultures exposed to MSH demonstrate equivalent increases in intracellular levels of cAMP. Further, neither theophylline (1mM), dibutyryl cyclic AMP (10(-4)M), or prostaglandin E1 (10(-6)M) is effective in stimulating tyrosinase activity in melanoma cells cultured at densities exceeding 6 X 10(4) cells/cm2. This finding suggests that the decay of hormone responsiveness occurs at a cellular site distal to cAMP production. The decrease in tyrosinase stimulation by MSH as cell density increases is also apparently not the result of an increase in activity of any soluble inhibitor of the enzyme, for cytosol preparations from high-density cultures (10(5) cells/cm2) fail to inhibit tyrosinase activity in cell homogenates from low-density cultures treated with MSH.     

The effects of pro-opiomelanocortin peptides on cyclic AMP and tyrosinase in melanoma cells

Des-, mono-, and diacetylated melanotropin (des-, mono-, and di-Ac MSH, respectively) were compared for their dose-related effects on content of adenosine 3':5'-monophosphate (cAMP) and tyrosinase activity in the Cloudman S91 mouse melanoma tumor. Des-Ac MSH was more potent than the acetylated forms of MSH at increasing cellular levels of cAMP; mono- and di-Ac MSHs, however, were more potent than des-Ac MSH at elevating the activity of the enzyme, tyrosinase. Lysine-gamma1 MSH, a melanotropin from the amino terminus of pro-opiomelanocortin, exhibited slight stimulatory effects on tyrosinase and these actions were less than additive to those of mono-Ac MSH. Unlike their actions on amphibian skin-darkening or in mammalian behavior, neither beta-endorphin1-31 nor its derivatives, N-Ac-beta-endorphin1-27 or beta-endorphin30-31 (glycylglutamine), exhibited any influence on tyrosinase activity evoked by mono-Ac MSH in the tumor cells.     

Effects of the lethal yellow (Ay) mutation in mouse aggregation chimeras

The Ay allele is a recessive lethal mutation at the mouse agouti locus, which results in embryonic death around the time of implantation. In the heterozygous state, Ay produces several dominant pleiotropic effects, including an increase in weight gain and body length, a susceptibility to hepatic, pulmonary and mammary tumors, and a suppression of the agouti phenotype, which results in a yellow coat color. To investigate the cellular action of Ay with regard to its effects upon embryonic viability and adult-onset obesity, we generated a series of aggregation chimeras using embryos that differ in their agouti locus genotype. Embryos derived from Ay/a x Ay/a matings were aggregated with those derived from A/A x A/A matings, and genotypic identification of the resultant chimeras was accomplished using a molecular probe at the Emv-15 locus that distinguishes among the three different alleles, Ay, A, and a. Among 50 chimeras, 25 analyzed as liveborns and 25 as 9.5 day embryos, 29 were a/a in equilibrium A/A and 21 were Ay/a in equilibrium A/A. The absence of Ay/Ay in equilibrium A/A chimeras demonstrates that Ay/Ay cells cannot be rescued in a chimeric environment, and the relative deficiency of Ay/a in equilibrium A/A chimeras suggests that, under certain conditions, Ay heterozygosity may partially affect cell viability or proliferation. In the 25 liveborn chimeras, Ay/a in equilibrium A/A animals became obese as adults and a/a in equilibrium A/A animals did not. There was no correlation between genotypic proportions and rate of weight gain, which shows that, with regard to its effects on weight gain, Ay heterozygosity is cell non-autonomous.     

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells.

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O₂ oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from ¹⁴C/³H leucine studies suggest that MSH may cause increased $\text{de}$$\text{novo}$ synthesis of tyrosinase.