Differential regulation and polyadenylation of transferrin mRNA in Xenopus liver and oviduct.

Estrogen destabilizes transferrin mRNA in male Xenopus liver in the same manner as observed for albumin and gamma-fibrinogen. The present study examined estrogen regulation of transferrin gene expression in female Xenopus liver and oviduct. In female Xenopus liver estrogen causes the same enhanced degradation of transferrin mRNA from the cytoplasm as seen in males. In contrast, transferrin is induced 3- to 4-fold in both oviduct nuclear and cytoplasmic RNA. The similar increase in transferrin RNA in both preparations suggests a transcriptional mechanism is responsible for this stimulation. Therefore, transferrin expression is differentially regulated in these tissues by the same hormone. Previous experiments showed that Xenopus serum albumin mRNA has a very short (17 residue) poly(A) tail that may play a role in its hormone-regulated instability. Transferrin mRNA has a similarly short poly(A) tail in liver of both male and female Xenopus. Estrogen has no effect on transferrin polyadenylation in liver. Similarly short poly(A) is found on transferrin mRNA from estrogen-deprived oviducts in explant culture. However, addition of estradiol to the medium results in the appearance of a 50-200 nucleotide poly(A) concurrent with induction. Therefore, transferrin mRNA is differentially polyadenylated in Xenopus liver and oviduct. In the latter tissue polyadenylation is under hormonal control.

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation.

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.

Nuclear androgen receptor dynamics in testicular peritubular and Sertoli cells.

Nuclear androgen receptor dynamics were analyzed in peritubular cells and compared with those in cultured Sertoli cells. Nuclear receptors with a high affinity for [3H]dimethylnortestosterone (DMNT; mibolerone) exhibited equilibrium constants of 0.8 and 0.7 nmol, in Sertoli and peritubular cells, respectively. Time- and dose-dependent accumulation of nuclear bound receptors after exposure of whole cells to [3H]testosterone was similar for both cell types. Exogenously administered ligands demonstrated similar relative potencies as competitors with [3H]T for Sertoli and peritubular cell nuclear binding sites: DMNT greater than T greater than medroxyprogesterone acetate (MPA) greater than cyproterone acetate (CPA) tau hydroxyflutamide (OHF). Cells incubated with T or MPA showed increased nuclear androgen receptor concentrations compared to untreated controls, whereas those treated with CPA or OHF did not. These results demonstrate that the nuclear androgen receptor dynamics of peritubular cells are consistent with those of target cells. Since the dynamics are similar in Sertoli and peritubular cells, both cell types have the potential to respond to local androgen concentrations and may play important roles in androgen-dependent effects on seminiferous tubule function.

Increase in testicular androgen receptor during sexual maturation in the rat.

Androgen receptor concentration was measured by exchange with 3H-dimethylnortestosterone (DMNT) in cytosol and nuclear extracts from testes of rats 15-90 days of age. Dissociation kinetics verified the necessity of an extended incubation (86 h) for maximum exchange at 4 degrees C. Nuclear androgen receptor concentration per mg DNA decreased between 15 and 25 days of age, from 375 to 146 fmol per mg DNA, then increased to 584 fmol per mg DNA at 90 days. Testicular receptor content also increased between 25 and 90 days of age. Cytosol receptor concentration patterns were similar to nuclear androgen receptor patterns. The affinity of the receptor for the ligand did not change with age (mean Kd = 0.88 nM). No significant difference in androgen receptor concentration per cell was detected between cultured peritubular cells from animals 25 and 45 days of age. Androgen receptor concentrations in freshly isolated peritubular cells could not be determined. There also was no difference in receptor concentration per cell in a Leydig cell-enriched fraction from animals between 25 and 45 days of age. Although androgen receptor concentrations per Sertoli cell increased between 15 and 35 days of age, the increase in Leydig cell number over the same period probably accounted for approximately 75% of the increase in receptor per testis between 25 and 45 days of age.

Evidence for age-dependent changes in Sertoli cell androgen receptor concentration.

Cytosol and nuclear receptor concentrations in Sertoli cells isolated from the testes of 15-, 25-, and 35-day-old rats were measured using hydroxylapatite separation procedures. In these cells the mean Kd of the cytosol receptor for methyltrienolone (3H-R1881) ranged between 2.3 and 2.9 nM, and the concentration of cytosol androgen receptor per mg Sertoli cell DNA increased over the 15-to 35-day age interval. However, when the data were expressed per mg cytosol protein, no increase was observed. The increase in receptor concentration per mg DNA paralleled the increase in cytosol protein/DNA ratio. The concentration of androgen receptor per mg DNA in nuclear extracts also increased with age. Consequently, total Sertoli cell androgen receptor increases over the time interval in which meiosis is first completed in the testis.

Hormonal regulation of Sertoli cell function.

The Sertoli cell is clearly influenced, directly and indirectly, by hormones. Among these are FSH, T, insulin and Vitamin A, but others may also be involved. Mechanisms are still not well understood. The biochemical effects of these hormones can be divided into quantitative and qualitative influences, with the former predominating. Specific cellular and secretory proteins and metabolites are affected, in many cases by more than one hormone. Often these same functions are influenced by other factors in the environment of the testis as well. Hormonal responsiveness of the Sertoli cell is determined in part by the maturational state of the cell. Some secreted products bind to specific cell types in the testis and epididymis and may influence the process of spermatogenesis. However, detailed mechanisms are not known at the present time. Understanding Sertoli cell function at the biochemical level and its control by hormones is clearly of key importance in understanding the control of the spermatogenic process.

Cortisol and its possible role in regulation of sodium and potassium in human milk.

Cortisol (F), Na, and K were measured in human milk samples taken every 4 h during 28 24-h cycles in 19 subjects. F in milk, measured by radioimmunoassay of unchromatographed extracts, demonstrated a circadian variation similar to that of plasma F and was dexamethasone-suppressible. Values ranged from 220 +/- 41 (SE) ng/dl at 2300 h to 1,214 +/- 210 at 0700 h. When milk extracts were chromatographed, a similar circadian variation was seen, and F ranged from 87 +/- 27 (SE) ng/dl at 2300 h to 784 +/- 164 at 700 h. The morning values of F exceeded previous estimates of corticosteroid binding capacity in milk, indicating the possibility of significant levels of unbound F. The subjects were divided into three groups according to time postpartum: I, 3.5-6 wk; II, 8-18 wk, and III, 20-32 wk. In milk, normalized values of F (unchromatographed) and electrolytes (x/means) were compared, with x the value at time t and means the mean value for a 24-h cycle. A significant circadian variation was seen in milk for (x/means)Na, (x/means)K, (x/means)Na/K, and x/means)F. Correlation of (x/means)F with (x/means)Na 4 h later was negative in all three groups (r = -0.36 to -0.56, P less than 0.10-0.001) as was (x/means)F vs. (x/means)Na/K (r = -0.33 to -0.61, P less than 0.10-0.001); (x/means)F vs. (x/means)K 4 h later was positive (r = 0.38 to 0.52, P less than 0.05-0.001). Dexamethasone administration caused a significant decrease in milk means Na and an increase in meansK in all five subjects (P less than 0.01). Thus, milk Na and K concentrations appeared regulated by adrenal corticosteroids.

Diurnal and longitudinal variations in human milk sodium and potassium: implication for nutrition and physiology.

Human milk sodium ([Nal]) and potassium ([K)] concentrations were measured every 4 h for 24 h in 28 subjects 3.5 to 32 wk postpartum. A diurnal variation in milk Na was seen, which was reciprocal to K. Significant negative correlations between Na and K were seen in these periods: 3.5 to 6; 8 to 18, 20 to 32 wk postpartum (p less than 0.01 for each). The mean 24 h milk sodium concentrations (x(Na)) decreased between 3.5 and 18 wk postpartum (p less than 0.005 by paired t test). Changes in mean potassium (-XK) were not statistically significant. Diet apparently does not affect milk Na. Administration of a low Na diet: 10.8 +/- .9 (SD) mEq Na/day and 60 to 100 mEq K/day for 2 days did not change x(Na). But urinary Na decreased 7-fold as aldosterone increased 5-fold. No significant correlation was seen between 24 h Na excretion in urine and x(Na) in milk (n = 51). A significant positive correlation was seen between urinary K and -XK in milk (r = 0.36), p less than 0.001).