Reductions in hypothalamic Gfap expression, glial cells and α-tanycytes in lean and hypermetabolic Gnasxl-deficient mice

BACKGROUND: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Gαs, termed XLαs, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. RESULTS: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive α-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3(rd) ventricle, while β-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. CONCLUSION: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since α-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections

HIGH POTASSIUM CONCENTRATION IMPROVES PREIMPLANTATION DEVELOPMENT OF MOUSE EMBRYOS INVITRO

Embryo growth in vitro in culture media containing potassium (K) ions at the concentration found in common extracellular fluids proceeds at a lower pace than in vivo. Considering that oviductal fluid has an unusually high concentration of K, the effect of various concentrations of this ion on development of mouse embryos in vitro was investigated. Two-cell to 4-cell preimplantation mouse embryos were cultured in vitro for 47 hours in a medium in which NaCl was partially replaced by KCl at concentrations ranging from 4.7 to 60 mM. The number of cells per embryo increased in a dose-related fashion when the embryos were cultured in the presence of 4.7, 10, and 25 mM of K. Higher K concentrations were detrimental for development. Embryos developed in vitro under different concentrations of K were transferred to pseudopregnant recipient foster mothers as a test of viability. The highest rate of implantation was observed with embryos cultured in medium containing 25 mM K. The results indicate that a high concentration of K in the culture medium (25 mM), comparable to that found in the genital tract of the female mouse, is required for a rate of development in vitro similar to the one observed in vivo

EFFECT OF ESTRADIOL-17BETA AND PROGESTERONE ON OVIDUCTAL TRANSPORT AND EARLY DEVELOPMENT OF MOUSE EMBRYOS

Transport of embryos through the oviduct, cleavage rate and transformation of morulae to blastocysts, were delayed in females ovariectomized on day 2 of pregnancy. Estradiol-17.beta. in doses of 60 to 6000 pg/day for 3 days did not normalize the transport of embryos, but the transformation of morulae to blastocysts reached values near or equal to those of the controls, in spite of a lowered rate of cleavage. Progesterone at a dose of 100 .mu.g/day, resulted in normal transport, rate of cleavage and rate of differentiation. Treatment with both hormones had synergistic effects on transport and the rate of cleavage and differentiation. Ovarian hormones are evidently the controlling factors for these processes in early pregnancy

EFFECT OF POST-COITAL ESTRADIOL TREATMENT UPON TRANSPORT, GROWTH, DIFFERENTIATION AND VIABILITY OF PRE-IMPLANTATION MOUSE EMBRYOS

The relative contribution of ovum transport anomalies and alterations of various developmental parameters upon the contraceptive effect of postcoital estrogen treatment was studied in mice. Females received a single s.c. injection from 0.001-100 .mu.g of estradiol or vehicle on day 1 of pregnancy and were sacrificed 28, 52 or 76 h after treatment to determine the location and development of embryos in the genital tract, or on day 12 of pregnancy to assess the number of viable implanted embryos. Preimplantation embryos were classified according to developmental parameters into normal, retarded or arrested. Pregnancy was completely suppressed by 10 and 100 .mu.g of estradiol. A partial and dose related block of pregnancy was observed with 0.01-1 .mu.g of estradiol. Treatment with these doses was associated with delayed oviductal transport, decreased cleavage rate, inhibition of blastulation and arrested development. The number of implanted embryos recorded on day 12 coincided with the number of normal embryos observed at 76 h, regardless of their location and numbers of cells. Statistically significant but minor decreases in the cleavage rate and transitory oviductal retention caused by estradiol neither prevented implantation nor affected subsequent viability of mouse embryos. The inhibition of pregnancy that follows postcoital administration of estradiol is associated with a variety of effects upon embryo transport and preimplantation development, which appear in different combinations with each dose and contribute unequally to its interceptive effect