Theresa C. Bienieki

The Developmental Fate of Melanocytes in Murine Skin

In the hair-covered general body skin of adult pigmented mice, melanogenically-active melanocytes are largely restricted to the bulbs of growing hair follicles. After cutaneous applications of appropriate chemical agents or exposure to ultraviolet light (UV), numerous melanotic melanocytes are found at the dermoepidermal junction of the interfollicular (superficial) epidermis and in the upper external root sheaths of hair follicles [6, 15 to 18]. These findings suggest that an extensive system of inactive “precursor” melanocytes forms an integral part of the epidermis in the hairy skin of adult mice. The melanocytes of this system although programmed to produce melanin granules and to discharge them into epidermal cells must await the application of an appropriate stimulus [13].

MODULATION OF MITOGEN-INDEPENDENT HEPATOCYTE PROLIFERATION DURING THE PERINATAL PERIOD IN THE RAT

SummaryLate gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2′-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor α (TGFα), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.

The Relationship between Differentiation and Proliferation in Late Gestation Fetal Rat Hepatocytes

Hepatocyte proliferation and differentiation occur simultaneously during late mammalian gestation. We hypothesized that regulation of hepatocyte growth and differentiation would be coordinated in late gestation fetal hepatocyte cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for α-fetoprotein (AFP), three markers of enzymic differentiation (glucokinase [GK], phosphoenolpyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), and a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was assessed using immunocytochemical detection of proliferating cell nuclear antigen (PCNA) or 5-bromo-2′-deoxy-uridine (BrdU) incorporation into DNA. Fetal hepatocyte cultures consisted of a heterogeneous population of cells, slightly more than half of which were proliferative under defined, growth factor-free conditions. These cultures were heterogeneous for AFP expression. There was no correlation between the expression of AFP and PCNA or AFP and S-phase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CAM. In addition, the differentiation status of cultured fetal hepatocytes was unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor α (TGFα), and hepatocyte growth factor (HGF). Finally, absence of any correlation between proliferation and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results indicate that the developmental program governing differentiation of late gestation fetal rat hepatocytes is independent from mechanisms controlling proliferation.

Effect of Altered Copper Metabolism Induced by Mottled Alleles and Diet on Mouse Tyrosinase

Summary An analysis of tyrosinase activity in extracts of hair bulbs from mice bearing alleles at the mottled locus, Mobr (brindled), and Moblo (blotchy), indicates that reduced melanogenesis in mottled mice is associated with restricted availability of copper. Although tyrosinase activity in 35,000g supernatants of hair-bulb homogenates of mottled mice is reduced compared to that of nonmottled controls, addition of copper to the former but not the latter supernatants markedly increases tyrosinase activity as measured in polyacrylamide gel electropherograms (dopa as substrate) or by the Pomerantz radioassay (tyrosine as substrate). Comparable results were obtained when tyrosinase from dietarily copper-deficient hypopigmented mice was assayed by either method. In the electropherograms of dietarily and genetically copper-deficient hair-bulb supernatants, the enhancement of activity is selectively restricted to the T1 variety of tyrosinase. It is concluded that alleles at the mottled locus influence tyrosinase and consequently melanin pigmentation by acting on melanocytes to reduce copper levels below those necessary to produce a normal complement of functional tyrosinase molecules.

Effects of Maternal Starvation on Hepatocyte Proliferation in the Late Gestation Fetal Rat

Fetal growth retardation, a common end point for a variety of conditions affecting mother and fetus, is associated with reduced liver mass. We have performed studies to determine the mechanism for decreased liver mass in a maternal starvation model of fetal growth restriction in the rat. Pregnant dams were deprived of food for 48 h before delivery on embryonic day 19 (E19). Fetal body weight was not affected. However, fetal liver weight was reduced by approximately 15%. Immunostaining of fetal liver for proliferating cell nuclear antigen and flow cytometry on isolated fetal hepatocytes showed G1 cell cycle arrest in samples from starved dams. Based on our prior studies showing attenuated hepatic insulin signaling in the late gestation fetal rat, we tested the hypothesis that G1 arrest in our model might be due to altered nutrient signaling. Fetal plasma amino acid analyses showed no decrease in branched-chain amino acids, but arginine concentrations were decreased in fetuses of fasted mothers. Reduced arginine in E19 fetal hepatocyte culture media was associated with decreased DNA synthesis. Whereas levels of cyclins D and E were unchanged in fetal hepatocytes exposed to low arginine, cyclin E-dependent kinase activity was reduced. Low arginine also induced changes in the translational machinery, indicative of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin. Our results are consistent with the hypothesis that restricted nutrient availability signals to the hepatocyte cell cycle in fetuses of fasted mothers, thereby accounting for decreased hepatocyte proliferation and liver mass.

Action of Trypsin and Detergents on Tyrosinase of Normal and Malignant Melanocytes

Summary The T1 variety of tyrosinase is present in both particulate and soluble or readily solubilized forms in the pigmented hypodermis (hair bulbs) of C57BL mice and Harding-Passey mouse melanoma. Trypsin treatment of 35,000g supernatants containing the microsomal (small granule) fraction of gentle homogenates of hair bulbs and melanoma results in significantly increased T1 activity within polyacrylamide gels. Similar treatment of 100,000g supernatants results in a slight increase in T1 activity. Addition of Triton-X or DOC to 35,000g supernatants of hair bulb and melanoma homogenates followed by centrifugation at 100,000g results in a marked enhancement of T1 when the latter supernatants are treated with trypsin. In the absence of trypsin treatment, T1 activity is comparable to nondetergent-treated controls. A slow-moving dopa-reactive band (Ts) is found in electropherograms of the nontrypsinized 100,000g supernatants of detergent-treated 35,000g supernatants. It is absent in those treated with trypsin. The slow-moving enzyme appears to give rise to T1 molecules when eluted from acrylamide gels and even to a greater extent when elution is combined with trypsin treatment prior to reelectro-phoresis. In mammals, tyrosinase apparently is not derived by a proteolytic activation of protyrosinase.

Ultrastructural observations on DMBA-induced dermal hyperpigmentation and blue nevus-like tumors in the Mongolian gerbil.

Summary Electron microscopic observations were made on the dermal hypepigmentation and blue nevus-like tumors induced in the dorsal trunk skin of Mongolian gerbils by DMBA. Hyperpigmented dermis was also examined in gerbils treated with DMBA and croton oil. The dermal hyperpigmentation was characterized by numerous melanin-bearing melanocytes and macrophages. Some of the dermal melanocytes were loaded with fully melanized (Stage IV) melanosomes, whereas others contained both mature melanosomes and immature ones (Stages I-III). Some dermal melanocytes displayed giant melanosomes (“mac-romelanosomes”) along with melanosomes of normal size. Finally, occasional dermal melanocytes exhibited typical Stages I-III melanosomes as well as round Stage I-like melanosomes in which melanin deposition occurred prematurely on the vesicular and filamentous matrices. The macrophages of hyperpigmented dermis contained groups of melanosomes segregated within membrane-limited secondary lysosomes. Paralleling the diversity in melanosome formation within the dermis, examination of DMBA-induced blue nevus-like tumors indicated that melanosomes were formed (1) in the standard manner with melanin deposited on a matrix of aligned filaments within a membrane-limited vacuole, and (2) by melanin deposition on a disorganized matrix of nonaligned filaments and/or mi-crovesicular bodies within a membrane-limited vacuole. Melanosomes formed via both pathways were subsequently grouped within autophagosomes and degraded therein.

Lactate dehydrogenase isozymes of mouse epidermis.

Five isozymes of LDH are demonstrable in the epidermis of the ear pinnae, hind feet, trunk dorsa, and tails of adult C57BL, C57HR, and C3HB mice by polyacrylamide gel electrophoresis. LDH-5 activity predominates in electropherograms. The ratio of LDH-1 to LDH-5 is greater in the epidermis of ear pinna and trunk dorsum than in that of tail and hind foot. The region-specific patterns of epidermal LDH isozymes are not correlated with melanin pigmentation or ‘hairiness’ of the skin.

STUDIES ON THE ONTOGENY OF FETAL HEPATOCYTE (FH) PROLIFERATION DURING LATE GESTATION IN THE RAT. |[dagger]| 341

We have found that late gestation rat FHs can proliferate in vitro in the absence of added mitogens or serum. However, this capacity varies with the gestational age at which the FHs are isolated: A gradual decline occurs as term approaches such that term FHs are non-proliferative in culture under these mitogen-free conditions. The present studies were undertaken to characterize the FH growth arrest which occurs at term. Primary cultures of FHs from day 19 of gestation (E19) showed a gradual decline in DNA synthesis (3H-thymidine incorporation) from very high levels during the first 24 hr in culture to low levels by 72 hr. In contrast, term (E21) FHs synthesized little DNA over the first 24 hr and showed a marked increase by 48 to 72 hr. In addition, E21 FH DNA synthesis was potentiated by Transforming Growth Factor α (TGFα) whereas E19 FH DNA synthesis was not. These findings correlated with immunocytochemical staining for Proliferating Cell Nuclear Antigen (PCNA). E19 FHs were PCNA positive immediately after plating and this persisted over 48 hr. E21 FHs showed a synchronized pattern of PCNA staining which was cytoplasmic at 12 hr and nucleolar at 24 to 48 hr. Intense, diffuse nuclear expression appeared for a brief period at 60 hr. Additional studies showed that: (a) E21 fetal liver has a high level of c-myc expression even though the cells are not proliferating; (b) The mitogenic Jun kinase signaling pathway is equally active at E19 and E21; (c) The cell cycle kinase, cdc2, is expressed at term but its inactive forms predominate. These results indicate that E21 FHs are growth arrested in vivo and that culturing releases them from inhibitory influences, resulting in synchronized proliferation in vitro.