We have studied contributions to hematopoiesis of genetically distinct stem cell populations in allophenic mice. Chimeras were made by aggregating embryos of inbred strains known to differ with respect to stem cell population kinetics. One partner strain (DBA/2) has previously been shown to normally have a stem cell (CFU-S) population of which 24% are in S-phase of the cell cycle, whereas the homologous population of the other partner strain (C57BL/6) was characterized by having only 2.6% in cycle (7). Contributions of the chimeric stem cell population to mature blood cell pools were studied throughout the life of the mice and intrinsic differences in stem cell function and aging were reflected in dynamic patterns of blood cell composition. The DBA/2 stem cell population was eclipsed by stem cells of the C57BL/6 genotype and, after 1.5-3 yr, the hemato-lymphoid composition of 22 of 27 mice studied for this long had shifted by at least 25 percentage points toward the C57BL/6 genotype. 8 of the 27 had hematolymphoid populations solely of C57BL/6 origin. To test whether or not a population of stem cells with an inherently higher cycling rate (DBA/2) might have a competitive advantage during repopulation, we engrafted allophenic marrow into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. DBA/2 hematopoiesis was predominant early, far outstripping its representation in the marrow graft. Perhaps as a consequence of inherently greater DBA/2 stem cell proliferation, the populations of developmentally more restricted precursor populations (CFU-E, BFU-E, CFU-GM, CFU-GEMM) showed an overwhelming DBA/2 bias in the first 2-3 mo after engraftment. However, as in the allophenic mice themselves during the aging process, the C57BL/6 genotypic representation was ascendant over the subsequent months. The shift toward C57BL/6 genotype was first documented in the marrow and spleen precursor cell populations and was subsequently reflected in the circulating, mature blood cells. Bone marrow-derived stromal cell cultures from engrafted mice were studied and genotypic analyses showed donor representation in stromal cell populations that reflected donor hematopoietic contributions in the same recipient. Results from these studies involving two in vivo settings (allophenic mice and engraftment by allophenic marrow) are consistent with the notion that a cell autonomous difference in stem cell proliferation confers on one population a competitive repopulating advantage, but at the expense of longevity.

During the aging process of males, testosterone biosynthesis declines in testicular Leydig cells resulting in decreases in various physiological functions. To explore the possibility of delaying the decline using food supplements, we have studied steroidogenic effects of a natural flavonoid, chrysin, in mouse Leydig cells. Chrysin dramatically increased cyclic AMP (cAMP)-induced steroidogenesis in MA-10 mouse Leydig tumor cells. This result was confirmed using Leydig cells isolated from mouse testes. The steroidogenic effect of chrysin is not associated with an increase in expression of the P450 side-chain cleavage enzyme, required for the conversion of cholesterol to pregnenolone. In addition, when 22(R)hydroxylcholesterol was used as a substrate, chrysin induced a non-significant increase in steroid hormone, suggesting that the majority of the observed increase in steroidogenesis was due to the increased supply of substrate cholesterol. These observations were corroborated by showing that chrysin induced a marked increase in the expression of steroidogenic acute regulatory (StAR) protein, the factor that controls mitochondrial cholesterol transfer. Also, chrysin significantly increased StAR promoter activity and StAR mRNA level. Further studies indicated that this compound depressed expression of DAX-1, a repressor in StAR gene transcription. In the absence of cAMP, chrysin did not increase steroidogenesis. However, when a sub-threshold level of cAMP was used, StAR protein and steroid hormone were increased by chrysin to the levels seen with maximal stimulation of cAMP. These results suggest that while chrysin itself is unable to induce StAR gene expression and steroidogenesis, it appears to function by increasing the sensitivity of Leydig cells to cAMP stimulation.

Testicular macrophages secrete 25-hydroxycholesterol, which can be converted to testosterone by neighboring Leydig cells. The purposes of the present studies were to determine the mode of production of this oxysterol and its long-term effects on Leydig cells. Because oxysterols are produced both enzymatically and by auto-oxidation, we first determined if testicular macrophages possess cholesterol 25-hydroxylase mRNA and/or if macrophage-secreted products oxidize cholesterol extracellularly. Rat testicular macrophages had 25-hydroxylase mRNA and converted 14C-cholesterol to 14C-25-hydroxycholesterol; however, radiolabeled cholesterol was not converted to 25-hydroxycholesterol when incubated with medium previously exposed to testicular macrophages. Exposure of Leydig cells to 10 microg/ml of 25-hydroxycholesterol, a dose within the range known to result in high basal production of testosterone when tested from 1 to 6 h, completely abolished LH responsiveness after 2 days of treatment. Because 25-hydroxycholesterol is toxic to many cell types at 1-5 microg/ml, we also studied its influence on Leydig cells during 4 days in culture using a wide range of doses. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol, with no cells dying at 10 microg/ml and only 50% of cells affected at 100 microg/ml after 2 days of treatment. Similar conditions resulted in 100% death of a control lymphocyte cell line. These results demonstrate that 1) testicular macrophages have mRNA for cholesterol 25-hydroxylase and can convert cholesterol into 25-hydroxycholesterol, 2) macrophage-conditioned medium is not capable of auto-oxidation of cholesterol, 3) Leydig cells are highly resistant to the cytotoxic influences of 25-hydroxycholesterol, and 4) long-term treatment with high doses of 25-hydroxycholesterol results in loss of LH responsiveness. These results support the concept that testicular macrophages enzymatically produce 25-hydroxycholesterol that not only is metabolized to testosterone by Leydig cells when present at putative physiological levels but also may exert inhibitory influences on Leydig cells when present for extended periods at very high concentrations that may occur under pathological conditions.

The carcinostatic activities of selenium (Se) compounds have been shown to be composition and concentration dependent. Several studies have indicated that the ratios between glutathione (GSH) and Se may play an important role in Se catalysis and toxicity. The present study examined the catalytic effect of three selenium compounds on GSH oxidation using lucigenin-dependent chemiluminescence (CL) as an indirect measure of superoxide generation. Various GSH:Se ratios were assayed for the glutathione oxidase activity of selenite, selenocystamine and diselenodipropionic acid. CL emitted from the reaction of selenite with GSH increased more rapidly and was greater than those from the diselenides, but the diselenide CL reactions were sustainable. Both selenite- and diselenide-induced CL were markedly suppressed by superoxide dismutase (SOD). Iodoacetic acid (IAc) effectively inhibited CL generated from selenite-, selenocystamine- and diselenodipropionic acid-catalyzed GSH oxidation. These results suggest that GSH oxidation catalyzed by selenite, and the diselenides selenocystamine and diselenodipropionic acid, generated the superoxide radical in which the CL was inhibited by SOD. Furthermore, CL inhibition by IAc suggests that the catalytic species producing superoxide were the GSSe(-) or RSe(-) anion. This redox chemistry may be responsible for selenite and organoselenium toxicity and apoptosis, making possible the design and synthesis of organoselenium-containing pharmaceuticals.

The science supporting the Dietary Reference Intakes (DRI) for phosphorus, magnesium, vitamin D, and fluoride was examined in this review. Along with the previous article on calcium in this series both of these reviews represent all the DRI for nutrients considered essential for bone metabolism and health, as reported in the Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride (Institute of Medicine, Food and Nutrition Board (FNB), 1997). The Recomended Dietary Allowances (RDA) or adequate intake (AI), and the tolerable upper intake level (UL) were recommended for each of these essential nutrients. For adults and in the case of fluoride, for infants as well, UL were calculated since all of these nutrients have the potential for mild to detrimental side effects. Dietary intake data and controversies regarding the role these nutrients may play in other chronic diseases have also been discussed. Advances and controversies reported since the publication of the DRI for these nutrients were also addressed in this review. A recent Dietary Reference Intake Research Synthesis Workshop report identified an extensive range of suggested future research directions needed to improve our understanding of these bone-related nutrients and their contributions to human health.

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.

The bioavailability of selenium (Se) from veal, chicken, beef, pork, lamb, flounder, tuna, selenomethionine (SeMet), and sodium selenite was assessed in Se-deficient Fischer-344 rats. Se as veal, chicken, beef, pork, lamb, flounder, tuna, SeMet, and sodium selenite was added to torula yeast (TY) basal diets to comprise Se-inadequate (0.05 mg Se/kg) diets. Se as sodium selenite was added to a TY basal diet to comprise a Se-adequate (0.10 mg Se/kg), Se-control diet. The experimental diets were fed to weanling Fischer-344 rats that had been subjected to dietary Se depletion for 6 wk. After 9 wk of the dietary Se repletion, relative activity of liver glutathione peroxidase (GSHPx) from the different dietary groups compared with control rats (100%) was: flounder 106%, tuna 101%, pork 86%, sodium selenite 81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58%. Se from flounder was the most efficient at restoring Se concentrations in the liver and skeletal muscle. Se from sodium selenite, SeMet, beef, veal, chicken, pork, lamb, and tuna was not dietarily sufficient to restore liver and muscle Se after 9 wk of recovery following a 6-wk period of Se depletion.

Genetic markers of two general types have been used to assess the number of simultaneously productive stem cells in vivo, retrovirus markers and enzyme or hemoglobin variants. Use of the two techniques has led to different conclusions regarding stem-cell population organization, kinetics, and usage. To better understand this discrepancy, we have combined the two methods by retrovirally marking and transplanting stem cell populations of allophenic mice in which all tissues, including the hematopoietic system, are chimeric. Hematopoietic and lymphoid tissues of engrafted recipients were analyzed by Southern blotting to determine the number and extent of participation of individually marked stem cells. Genotypic chimerism of the same tissues was determined by quantitating electrophoretic variants of glucose phosphate isomerase. This procedure permitted the genotypic identification of individual stem-cell clones. The results demonstrate the participation of few pluripotent stem cells in the repopulation and maintenance of engrafted hematopoietic and lymphoid tissues. Furthermore, stem cells used during the period of early engraftment tended to be of one genotype (DBA/2), whereas stem cells used for long-term maintenance tended to be of the other, coexistent genotype (C57BL/6). We propose that this genotypic specificity reflects functional differences in stem-cell subpopulations and their relative prevalence in different mouse strains suggests a genetic component in the organization and usage of stem cells.