Oral polyamine administration modifies the ontogeny of hexose transporter gene expression in the postnatal rat intestine.

Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na(+)-K(+)-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, alpha(1)- and beta(1)-subunits of Na(+)-K(+)-ATPase, and ODC. Postnatal rats fed 6 mumol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na(+)-K(+)-ATPase activity and alpha(1)- and beta(1)-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.

Effect of maternal serum on viability and function of early human placental explants.

Fetal bovine serum (FBS) is frequently used to supplement chemically defined media such as Ham's F10 when studying placental explant cultures. However in vitro production of hormones is usually declining by the 2nd or 3rd day and is short-lived (7 to 10 days). In this study we explored the use of human maternal serum (HMS) from early gestation as the medium supplement to Ham's F10. Early placental hormone production was compared using two concentrations of FBS and HMS. On Day 3 of incubation, progesterone production in 10% HMS was 12-fold increased over that in 10% FBS, estradiol production was increased 10-fold, and beta hCG production more than 3-fold. When the serum concentrations were increased to 40%, the results in all cases were similar to those at 10%. Preliminary characterization studies revealed that the stimulatory activity of HMS is heat-labile, neither extractable into organic solvent (diethyl ether) nor dialyzable, suggesting that it is protein in nature. In a long-term incubation, compared with FBS (7 days), HMS permitted survival of culture up to 30 days, judged both histologically and biochemically. We conclude that HMS provides substance(s), probably protein in nature, not present in FBS or non-pregnant human serum, which are important for human placental viability and function in vivo.

Effect of maternal serum on viability and function of early human placental explants.

Fetal bovine serum (FBS) is frequently used to supplement chemically defined media such as Ham's F10 when studying placental explant cultures. However in vitro production of hormones is usually declining by the 2nd or 3rd day and is short-lived (7 to 10 days). In this study we explored the use of human maternal serum (HMS) from early gestation as the medium supplement to Ham's F10. Early placental hormone production was compared using two concentrations of FBS and HMS. On Day 3 of incubation, progesterone production in 10% HMS was 12-fold increased over that in 10% FBS, estradiol production was increased 10-fold, and ßhCG production more than 3-fold. When the serum concentrations were increased to 40%, the results in all cases were similar to those at 10%. Preliminary characterization studies revealed that the stimulatory activity of HMS is heat-labile, neither extractable into organic solvent (diethyl ether) nor dialyzable, suggesting that it is protein in nature. In a long-term incubation, compared with FBS (7 days), HMS permitted survival of culture up to 30 days, judged both histologically and biochemically. We conclude that HMS provides substance(s), probably protein in nature, not present in FBS or non-pregnant human serum, which are important for human placental viability and function in vivo.

Influence of 19-nortestosterone and androgens on progesterone biosynthetic enzymes in early human placental explants.

We recently showed that the production of progesterone (P4) in human placental explant culture from early gestation is enhanced by treatment with 19-nortestosterone (19-NT) or with certain androgens, namely androstenedione (A-dione), 5 alpha-androstane-3 alpha,17 beta diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta diol (3 beta-diol). This stimulation of P4 was explored further in this study. There was little metabolism of radioactive P4 when incubated for 24 h in the presence or absence of these steroids. The role of different steroids in the regulation of P450 cholesterol side-chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was evaluated by measuring the conversion of P4 derived from unlabelled 25-hydroxycholesterol and from labelled pregnenolone, respectively. The results showed that 19-NT, A-dione and 3 alpha-diol stimulated P450scc activity; however, 3 beta-diol was ineffective. While 19-NT and 3 beta-diol enhanced the bioconversion of pregnenolone to P4, A-dione and 3 alpha-diol were without effect. The initial rapid stimulation of P4 by 19-NT within 2 h of incubation was not blocked by concurrent treatment with cycloheximide (CH). However, after incubation for 24 h, 70% of the 19-NT-stimulated P4 was abolished by CH. During the same incubation period, P4 stimulation by A-dione, 3 alpha- and 3 beta-diol were completely blocked by treatment with CH. Thus our observations suggest that 19-NT-stimulated P4 accumulation is due to the combined effects on P450scc and 3 beta-HSD enzyme activities. A-dione and 3 alpha-diol increase biosynthesis of P4 by acting selectively on P450scc enzyme. However, the stimulatory action of 3 beta-diol on P4 is only at the level of 3 beta-HSD. Since CH blocks the stimulatory actions, the mechanism(s) by which androgens (A-dione, 3 alpha-diol and 3 beta-diol) and norandrogen (19-NT) augment the biosynthetic enzyme activities appears to be mediated by a process inhibited by CH. Since CH interference was absent during the initial rapid P4-stimulation by 19-NT, there may be a direct action of this steroid at the cellular level which is not dependent on new protein synthesis.

In vitro stimulation of placental progesterone production by 19-nortestosterone and C19 steroids in early human pregnancy.

To explore the regulatory mechanism at the critical period of the luteal-placental shift, the effects of various steroids and peptides on the production of progesterone by placental explants at 7-10 weeks were studied. Androstenedione increased progesterone production 3-fold at a concentration of 1 mumol and more than 20-fold at 18 mumol. 19-Nortestosterone (1-18 mumol) stimulated progesterone production 10- to 100-fold. 5 alpha-Androstane-3 beta,17 beta diol (1-18 mumol) stimulated progesterone production about 2- to 5-fold while its 3 alpha isomer (1-6 mumol) increased it 2-fold. Estrone, estradiol, and estriol up to a concentration of 30 mumol had no effect. Dehydroepiandrosterone sulfate (to 36 mumol), androst-5-ene-3 beta,17 beta diol (1-6 mumol), 5 beta-androstane-3 alpha,17 beta diol (1-6 mumol), and dihydrotestosterone (1-12 mumol) had no effect. Cortisol and dexamethasone (up to 12 mumol), hCG (20,000 IU/L), GnRH (4 mumol), and ACTH 1-24 (20 mumol) also had no effect. Thus, of all the compounds tested, only 19-nortestosterone and, to a lesser extent, androstenedione, 5 alpha-androstane-3 beta,17 beta diol, and 5 alpha-androstane-3 alpha,17 beta diol stimulated progesterone production in early pregnancy; at term, only 5 alpha-androstane-3 beta,17 beta diol was stimulatory. 19-Nortestosterone was found to be less efficiently aromatized compared to other androgens; since it is also known to be present in blood from pregnant women and thought to be made in the placenta, the stimulation observed may be a paracrine effect. These observations suggest that C18 and C19 steroids may be important in the regulation of progesterone synthesis by the human placenta in early pregnancy.