Embryo survival, and fetal and placental growth following elevation of maternal estradiol blood concentrations in the rat.

High doses of estrogens cause embryonic mortality, and fetal and placental growth retardation in rats. This study addresses the physiological relevance of such findings. Estradiol benzoate (EB), by s.c. injection, or estradiol-17beta (E2), delivered by a miniosmotic pump, raised maternal E2 concentrations from only slightly above control values to 5-fold. EB (1 microgram/day) over Days 6-13, 8-13, and 11-13, and continuous infusion of E2 (15 ng/h; Days 10-13) reduced fetal survival to 0%, 0%, 22%, and 75%, respectively. Single injections of EB showed that its lethal effect declined rapidly over Days 9 (44% survival) to 13 (90% survival). Embryos died within 48 h, but death was not due to luteal failure since progesterone levels were maintained and progesterone administered with EB did not reduce mortality. Administration of EB at 1 microgram/day (Days 14-21) or E2 at 40 ng/h (Days 13-16) retarded fetal and placental growth but did not affect survival. The rat embryo is highly sensitive to elevated maternal estradiol concentrations over much of gestation. The early lethal effect implies that endogenous E2 production is carefully regulated to maintain pregnancy; the latter growth-retarding effect suggests that E2 may have a role in the normal control of fetal growth.

Characterization of ganglioside associated with the thyrotrophin receptor.

The receptor protein for thyrotrophin (thyroid-stimulating hormone; TSH) is associated with a glycosphingolipid moiety. The protein belongs to the family of receptors that couple to guanine nucleotide binding proteins; the glycosphingolipid contains sialic acid and belongs to the family of gangliosides. This report defines the structure of the receptor ganglioside in the Fisher rat thyroid cell line (FRTL-5). Receptor protein was purified by TSH affinity chromatography from FRTL-5 cells, biosynthetically labelled with [3H]galactose and [3H]glucosamine, and resolved by SDS-PAGE. A single radiolabelled band of Mr approximately 80 kDa, corresponding to the predicted size of the cloned receptor, contained ganglioside. Gangliosides were extracted from unlabelled receptor protein after SDS-PAGE and probed on TLC plates with 125I-labelled Limax flavus agglutinin or the B subunit of cholera toxin, before and after digestion with Vibrio cholerae sialidase or beta-galactosidase. The TSH receptor (TSH-R) ganglioside belongs to the gangliotetraose family, having sialic acid attached to both galactose molecules. Its sialic acid is devoid of negative charge because of the formation of internal esterlactones. Its structure is lactonized N-acetylneuraminyl-(alpha 2-->3)galactosyl(beta 1-->3)-N-acetylgalactosaminyl(beta 1-->4)-[N-acetylneuraminyl(alpha 2-->3)]galactosyl(beta 1-->4)glucosyl(beta 1-->1)ceramide (GDla-lactone). Ganglioside lactones have not been previously described as components of thyroid cells. They are highly rigid and are more likely than their parent structures to serve as molecular recognition sites and elicit immunoreactivity. Identification of this unique ganglioside intimately associated with the TSH receptor implies that it has an integral role in receptor structure and function.

Overexpression of beta 2-microglobulin in transgenic mouse islet beta cells results in defective insulin secretion.

Overexpression of heavy chains of the class I major histocompatibility complex in islet beta cells of transgenic mice is known to induce nonimmune diabetes. We have now overexpressed the secretory protein beta 2-microglobulin in beta cells. Transgenic mice of one lineage had normal islets. Mice of another lineage did not become overtly diabetic but showed significant depletion of beta-cell insulin. When mice were made homozygous for the transgene locus, they developed diabetes. Introduction of the beta 2-microglobulin chain into class I heavy chain transgenic mice resulted in a significant improvement in their islet morphology and insulin content, and the female mice remained normoglycemic. These results suggest that different transgene molecules overexpressed in beta cells can cause islet dysfunction, though not necessarily overt diabetes, and that this effect is mediated by the level of transgene expression. Evidence is provided to show that beta-cell disruption by transgene overexpression occurs at the level of protein and involves a defect in insulin secretion.

A2B5-reactive ganglioside expression determines the differentiation stage and capacity of rat insulinoma (RIN) sublines.

We have generated rat insulinoma (RIN) sublines AlGh, m5F, A12, A13 and AhGh with increasing surface expression of the A2B5 ganglioside, a marker of differentiation. We asked whether the capacity of the sublines to differentiate was related to their stage of differentiation, as is characteristic of cells within the normal beta-cell lineage. To answer this, we measured the effect of the differentiation inducer sodium butyrate (NaB, 1 mM) on proliferation, insulin content, secretion and biosynthesis, and the expression of A2B5 and 3G5 gangliosides by the sublines. Six days after exposure to NaB, cell numbers/dish ranged from (1-3) x 10(6) compared to (4-6) x 10(6) in control cultures. By day 2, AlGh, m5F, A12, A13 and AhGh cells exposed to NaB contained 1.5-, 1.4-, 1.4-, 1.2- and 1.0-fold higher amounts of insulin, respectively, and by day 6, 3.6-, 2.3- and 1.0-fold higher, and 1.2- and 2.4-fold lower, amounts of insulin, respectively, than control cells. After 2 days, insulin secretion from AlGh, m5F, A12, A13 and AhGh cells was 1.7-, 1.0-, 1.5-, 1.0- and 1.0-fold higher, respectively, and the rate of (pro)insulin biosynthesis 1.7-, 2.3-, 1.3-, 1.0- and 1.0-fold higher, respectively, than control cells. After 6 days, A2B5 ganglioside expression was increased 3-, 1.9- and 2-fold on m5F, A12 and A13 cells, respectively, but was not significantly altered on AlGh and AhGh cells. 3G5 ganglioside expression was increased 1.5- and 8.4-fold, respectively, on AlGh and m5F cells, but was unaltered on A12, A13 and AhGh cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A2B5-reactive ganglioside expression is an index of differentiation in rat insulinoma (RIN) cells.

To investigate the relationship between surface ganglioside expression and pancreatic islet cell differentiation, we examined the function of five rat insulinoma (RIN-m5F) sublines A4, A6, A7, A10, and A12 selected for increased expression of A2B5-reactive gangliosides, as well as a subline AlGh, low in A2B5- but high in 3G5-reactive ganglioside expression. Class I major histocompatibility (MHC) protein expression was also measured in the sublines because of our previous finding that class I proteins were preferentially expressed on human insulinoma tissue compared with differentiated islet cells. In comparison with parental RIN-m5F cells, subline A12 displayed a 7.6-fold increase in A2B5 expression and a 3.4-fold increase in the number of A2B5 positive cells (81% vs. 24%). A2B5 expression was increased 1.3-, 5.4-, 5.4-, and 6.9-fold on A4, A6, A7, and A10 sublines, respectively. In contrast, AlGh cells, which had comparable A2B5 expression to parental cells, displayed a 2.9-fold increase in 3G5 expression and an 8-fold increase in the number of 3G5 positive cells (72% vs. 9%). Insulin secretion and content increased with increasing A2B5 expression. On day 2, secretion was 1.2-, 8-, 8-, 21-, and 18-fold higher and content 1.4-, 4-, 5-, 26-, and 33-fold higher for A4, A6, A7, A10, and A12 cells, respectively, compared to parental cells. There was a direct association between expression of A2B5 and the level of insulin messenger RNA (mRNA) in the sublines. Neither glucagon nor somatostatin was detected in any subline. The AlGh subline secreted and contained less insulin than parental cells. Fully differentiated adult rat islet cells, the majority of which are beta-cells, contained a lower number of 3G5 (12%) than A2B5 (57%) positive cells. Compared to parental cells, class I MHC proteins were decreased 4-fold on A12 cells, but increased 1.5-fold on AlGh cells. We conclude that, at least in RIN cells, the expression of A2B5-reactive ganglioside expression is associated directly, and class I MHC protein expression indirectly, with beta-cell differentiation.

Pancreatic islet A2B5- and 3G5-reactive gangliosides are markers of differentiation in rat insulinoma cells.

Rat insulinoma (RIN) cells, in comparison with adult islet cells, are relatively undifferentiated. They secrete low amounts of islet hormones, are unresponsive to glucose, and display pluripotency. A minority of RIN cells react with monoclonal antibodies A2B5 and 3G5 which recognize complex gangliosides on normal islet cells. In order to determine whether the expression of A2B5- or 3G5-reactive gangliosides is modulated during differentiation RIN cells were cultured with various concentrations of sodium butyrate (NaB), a known inducer of cellular differentiation. Expression of A2B5- and 3G5-reactive gangliosides was determined by indirect immunofluorescence and flow cytofluorimetry. NaB exposure resulted in a dose-dependent decrease in cell proliferation over 5 days of 1.5-, 2.9-, and 17.5-fold at 0.5, 1.0 and 3.0 mM, respectively, and a distinct change in cellular morphology. Cells exposed to NaB displayed prominent neurite-like projections. At 3 mM NaB, insulin secretion increased 7.9-fold and the percentage of cells expressing A2B5- and 3G5-reactive gangliosides increased by up to 4.4- and 5.5-fold, respectively. The expression of A2B5- or 3G5-reactive gangliosides per cell also increased, by 2.4- and 1.3-fold, respectively, at 3 mM NaB. These findings demonstrate that the expression of cell surface A2B5- and 3G5-reactive gangliosides is not static but increases with cell differentiation. NaB-treated RIN cells may serve as a model to study the role of gangliosides in the function and lineage relationships of islet cells.

A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells.

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.

Anchorage-independence as an index of proliferative potential and maturational age: a comparison of the growth of normal, primary explanted bovine granulosa cells in semisolid agar and in liquid culture.

When seeded at low-density, normal primary explanted granulosa cells will grow to form clones of functionally differentiated cells in both semisolid agar and in liquid culture. The anchorage-independent clonogenic granulosa cell differs from the anchorage-dependent granulosa cells detected in clonal liquid culture in a number of properties. Basal cloning efficiency in liquid culture is up to 50-fold higher than in agar culture. In serum supplemented medium (20% fetal calf serum) cloning efficiency in liquid culture is unaltered in the presence of added epidermal growth factor (EGF), whereas, agar cloning efficiency is augmented six-fold when cells are incubated under identical conditions. Cells derived from primary anchorage-independent clones, when dispersed and replated, will generate secondary anchorage-independent clones and anchorage-dependent liquid clones. On the other hand, although cells derived from parallel primary anchorage-dependent clones will also generate secondary anchorage-dependent clones, generation of secondary anchorage-independent clones is not detectable. These findings suggest that the anchorage-independent clonal agar assay may be detecting a developmentally earlier granulosa cell subpopulation than is detectable in the liquid culture assay.

Serial measurement of arterial plasma progesterone levels throughout gestation and parturition in individual rats.

Daily blood samples were taken from 6, chronically cannulated, fully conscious rats to measure plasma progesterone levels throughout gestation. Progesterone levels in individual rats fluctuated by up to 28 ng/ml per day, but tended to be consistently higher or lower than the group mean. The accuracy of predicting progesterone levels in individual rats from previous values was examined. Progesterone levels on day 7 of gestation were negatively correlated with foetal weights near term. There was little indication that high progesterone levels at any stage of gestation lead to increased foetal or placental weights. Progesterone levels on day 17 were positively correlated with the number of corpura lutea but there was little relationship between progesterone and either the number or total mass of the placentas. Serial blood samples taken from a second group of 6 rats at 2 hourly intervals showed that the time between the major fall in progesterone levels to below 12 ng/ml and the onset of parturition was relatively constant (varying by only 8 h) despite a 29 h range in the total length of gestation.