Tetsuo Noumura

Age-associated changes in plasma testosterone levels in male mice and their relation to social dominance or subordinance

Abstract Plasma testosterone (T) levels were determined in male mice of the CD-1 (ICR) strain from 30 to 680 days of age. The mean plasma T was 5.2 ± 1.0 ng/ml for adult male mice (70–400 days old, 102 mice) and 1.8 ± 0.9 ng/ml for old males (450–680 days old, 11 mice). Through 140 determinations, the highest T value obtained was 52.3 ng/ml in an 80-day-old individual while the lowest was 0.08 ng/ml in a 680-day-old animal. It was found that plasma T was significantly decreased in the old age, although individual variation in T levels was considerable. On the other hand, social dominance or subordinance was examined among male mice in each cage and its relation to plasma T was investigated. Dominance or subordinance was verified by frequency of chases or attacks delivered or received, or number of fights won or lost. It was observed that dominant-subordinate relationships were present in 6 out of 10 cages and that the dominant individual had a significantly higher T level than the subordinate male; the mean T level was 10.5 ± 2.5 ng/ml for the dominant individuals vs 2.2 ± 0.8 ng/ml for the subordinate ones. Marked individual variation in plasma T titers in male mice seems to be related to the dominance/subordinance rank within a group.

Effects of various steroids on in vitro lifespan and cell growth of human fetal lung fibroblasts (WI-38)

Human fetal lung fibroblasts, WI-38, were cultivated in a medium containing various steroids. A dose-response curve constructed by counting the number of cells on day 16, or at regular intervals, showed that glucocorticoids (hydrocortisone and cortisone) and mineralocorticoids (aldosterone and deoxycorticosterone) caused an increase in cell density when added at physiological concentrations or higher. On the other hand, androgens (testosterone, dihydrotestosterone, and dehydroepiandrosterone), estrogen (17 beta-estradiol), and progesterone caused a decrease in cell density at higher concentrations (5 micrograms/ml, or more) although these had no effect on cell density at concentrations lower than 0.5 micrograms/ml. With cells grown from population doubling 31 in a medium containing steroids, it was shown that hydrocortisone extended the in vitro lifespan of WI-38 cells at concentrations of both 5 micrograms/ml and 0.5 micrograms/ml, while dehydroepiandrosterone, 17 beta-estradiol, and progesterone caused a shortening in lifespan at only 5 micrograms/ml. These results suggest that there is a direct relationship between the effects of steroids on cell growth and the lifespan of human fetal lung fibroblasts.

Sexually dimorphic and laterally asymmetric development of the embryonic duck syrinx: effect of estrogen on in vitro cell proliferation and chondrogenesis

The syrinx, the vocal organ in birds, shows sexual dimorphism in the duck, Anas platyrhynchos. At the cellular level, to examine the role of estrogen in sexually dimorphic and laterally asymmetric development of duck syrinx, cells dissociated from the right and left halves of sexually monomorphic and primitive masculine syrinxes from 10 2/3-day embryos of male and female ducks were cultured by the micromass culture method, with or without estrogen. In the absence of estrogen, primary cell cultures from either side of male syrinx revealed identical abilities in both cell proliferation and chondrogenesis. In the female cell cultures, the right- and left-side cells proliferated equally but the right-side cells accumulated a larger amount of sulfated proteoglycans than the left-side cells did. Both proliferation and chondrogenesis in the male cell cultures were more active than those in the female cell cultures. Estradiol inhibited significantly cell proliferation as well as chondrogenesis in cell cultures from either side of female syrinx. Cultures from right-side cells of male syrinx were less inhibited by estradiol in cell proliferation and especially in chondrogenesis than the other three cultures. Generally, in both sexes the left-side cells of syrinx were more responsive to estrogen than the right-side cells in diminishing proliferation and chondrogenesis. The present results suggest that estrogen inhibits both cell proliferation and chondrogenesis in the female syrinx and that this process may contribute to the development of sexual dimorphism in the duck syrinx.

The heterogeneity of human fibroblasts as determined from the effects of hydrocortisone on cell growth and specific dexamethasone binding

To elucidate the heterogeneity of human fibroblasts from lung and skin, the effects of hydrocortisone on cell proliferation and the specific dexamethasone binding to cells were studied. Hydrocortisone at physiological concentrations stimulated the proliferation in three strains of human fetal lung fibroblasts and inhibited it in two strains. There are two kinds of fibroblasts in the human fetal lung in addition to the human skin fibroblasts reported previously. Dexamethasone-binding experiments showed that human fibroblasts may be classified into two groups with respect to the dissociation constant (Kd) of the binding reaction. The heterogeneity of human fibroblasts shown by Kd could not be correlated to classification on the basis of the effects of hydrocortisone on cell proliferation. The differences in Kd for the binding reactions suggest differences in donor tissues from which human fibroblasts are derived.

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen☆

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.

Age-related changes in the mitogenic activity of heparin-binding growth factors in rat sera

Sera from rats of either sex and different ages were examined for their ability to stimulate DNA synthesis in BALB/c 3T3 cells. The activity levels of sera from male and female rats were almost the same, with age-related changes in activity also being quite similar. Activity was considerably higher in infant rats (1-month-old), but then, at a young age (6-7 months), decreased drastically for male rats, but not significantly for female rats. It increased again in middle-aged rats (12-13 months old) and was maintained at the same level toward old age (24-26 months old) for both sexes. In order to determine what kinds of growth factors were responsible for these changes, we carried out heparin affinity chromatography on the sera of male rats. Four peaks were obtained for all sera, with individual peaks exhibiting specific age-related changes in activity. Among them a peak which was eluted at 1.1 M NaCl had very high activity. It showed a similar age-related change to that of the whole sera, except for a significant increase at old age, and the factor(s) included in the peak was found to be derived from platelets. These results suggested that the factor(s) in the peak was responsible for maintaining serum mitogenic activity at an old age. The experiments undertaken to characterize this factor suggested that it is a novel one.

Growth hormone-releasing factor (GRF) suppresses the in vitro proliferation of mammotrophs from the adult rat.

In order to examine the hypothalamic control of acidophilic proliferation, anterior pituitary cells from adult female rats were cultured with or without rat growth hormone-releasing factor fragment 1-29 (GRF-29). Changes in the numbers of mammotrophs and somatotrophs during culture were measured by immunocytochemical staining. The addition of GRF suppressed the increase in the number of mammotrophs even at the very low concentration of 10(-12) M. The number of somatotrophs increased in the medium containing GRF. The increase in mammotroph number was blocked by cytosine arabinoside, a mitotic inhibitor. GRF had no effect on the in vitro proliferation of fibroblasts. These results indicate the important role of hypothalamic GRF in the differential growth and secretion of the acidophils in vivo.

Effects of Inhibin on Rat Gonadal Differentiation and Development In Vitro

Abstract Previously we examined that inhibin-&agr; subunit, transforming growth factor-&bgr;1 (TGF-&bgr;1) and epidermal growth factor (EGF) were expressed in sex-, cell- and stage-specific manners in perinatal rat gonads. To clarify effects of these growth factors on the rat gonadal differentiation and development, indifferent gonadal primordia with mesonephric tubules on gestational day 13 were cultured in vitro for 4 days in serum-free CMRL-1066 medium with inhibin, TGF-&bgr;1, EGF, anti-sera against these growth factors, testosterone or estradiol-17&bgr;, and then morphologically examined with reference to seminiferous tubule formation, germ cell division, Wolffian and Müllerian duct development. In male gonads, anti-inhibin-&agr; serum suppressed the seminiferous tubule formation but inhibin, TGF-&bgr;1, EGF or steroid hormones did not affect on the tubule formation, germ cell proliferation nor gonoduct development. Seminiferous tubules in male gonads cultured in the medium containing anti-inhibin-&agr; serum were incomplete and irregular in shape. On the other hand, in female gonads, inhibin suppressed the germ cell division and anti-inhibin-&agr; serum led to the necrosis of germ cells, but other factors affected to neither sex cord formation nor germ cell division. Testosterone and estradiol-17&bgr; stimulated female Wolffian and Müllerian duct development, respectively. These results indicate that inhibin induces the seminiferous tubule formation and suppresses the female germ cell division in developing rat gonads in vitro.

Laterally Asymmetric Development of Duck Syrinx: Inhibition of Growth and Chondrogenesis of the Right Syringeal Half by the Left Half

For determination of whether position‐related tissue interaction is involved in the laterally asymmetric development of the male duck syrinx, a cartilaginous vocal organ, the syrinxes from 10‐day embryos were cut into right and left halves and cultured organotypically. The lateral halves developed equally when cultured separately, but their developments were unequal when they were cultured together in the same culture medium: that is, the right half developed less than the left half in terms of increase in size and chondrogenesis. When the right half from a normal or estrogen‐treated embryo was cultured with the left half from a normal embryo, it accumulated less sulfated proteoglycans than when it was cultured with a left half from an estrogen‐treated embryo. These results suggest that a soluble inhibitor that is susceptible to estrogen in the left half of the male syrinx inhibits the intrinsic increase in size and chondrogenesis of the right half and plays an important role in the laterally asymmetric development of the male duck syrinx.