Gunn & Gould (1958) introduced evidence of inherent testicular rhythms in the laboratory rat. They reported that the uptake of radioactively labelled zinc by the dorsolateral prostate (DLP) during the span of 1 year shows two distinct maxima, in February and in June. Further support of this concept came from studies of androgen biosynthesis by the rat testis in vitro (Ellis, 1970). Recent investigations in this laboratory reveal additional, more direct, information concerning rhythmic endocrine performance and also the possibility of a rhythm in gametogenic function of the rat testis. Male Sprague—Dawley rats, 4 weeks of age, were housed individually in suspended wire-mesh cages at room temperature (21 ± 1 °C) and relative humidity (45–65%). The daily lighting cycle was 12 h light: 12 h darkness (lights on 06·00–18·00 h) and the animals received food and water ad libitum. Functions of the testes were subsequently examined with groups of six

Abstract

Oxytocin (OT) is a neurohypophysial hormone with potent stimulating activity of the pregnant uterus, but its physiological role in parturition is still unclear. Recently, OT was found to be synthesized in the pregnant uterus, indicating that OT originating from the uterus, not from the posterior pituitary gland, may trigger the onset of labour. In order to define the factors responsible for the induction of uterine OT, the effect of ovarian steroid hormones and conceptus on the induction of OT mRNA in the rat uterus was examined by Northern and dot blot hybridization analysis. OT mRNA in the uterus started to increase on day 14 of pregnancy and showed very high levels at the time of parturition. Uterine OT mRNA was not altered by any steroid treatment, oestradiol-17β (0·2 μg), progesterone (4 mg) or both in combination, for 6 days. The gravid horn of the uterus had 3·6-fold as much OT mRNA as the non-gravid horn on day 21 of pregnancy in hemipregnant rats with one ligated oviduct. The ovarian steroid hormones could not induce accumulation of OT mRNA in the uterus of ovariectomized rats, at least under the conditions used, but the presence of a conceptus may be critical for the very high levels of OT mRNA.

Journal of Endocrinology (1995) 146, 81–85

ABSTRACT

Glucocorticoids are known to regulate the contractility of vascular smooth muscle by increasing its response to noradrenaline. The molecular mechanisms for achieving this remain unclear. Recent results in our laboratory have demonstrated that glucocorticoids affect both α₁-adrenoceptor number and coupling to G proteins. Whether this leads to an increase in second-messenger production has to be established. The present experiments, therefore, report the effects of dexamethasone on inositol polyphosphate production in vascular smooth muscle cells in culture. Noradrenaline induced the release of inositol polyphosphates from prelabelled [³H]inositol phosphoinositides in the membrane in a dose-dependent manner. The concentration of noradrenaline which caused half-maximal response was 1·26 μmol/l. Prazosin inhibited noradrenaline-induced inositol monophosphate formation to 10·26 ± 3·67% (mean ± s.e.m.; P < 0·01, n = 5) of control value whereas yohimbine reduced it to only 61·74 ± 11·82% (P < 0·05, n = 5), suggesting an action primarily through α₁-adrenergic receptors. Dexamethasone (100 nmol/l, 48 h) enhanced noradrenaline-induced inositol monophosphate, bisphosphate and trisphosphate formation up to twofold (P < 0·001, n = 5). The enhancement of the response occurred despite the fact that dexamethasone reduced [³H]inositol prelabelling of membrane phosphoinositides by 49·5 ± 9·9% (P < 0·05, n = 3). The present results suggest that the potential action of glucocorticoids on vascular smooth muscle contractility is, at least in part, through controlling α₁-adrenoceptor-mediated second-messenger production.

Journal of Endocrinology (1992) 133, 405–411

Abstract

Caprine chorion, allantois and amnion from days 23, 28, 35, 39 and 45, and yolk sac from day 23 of pregnancy were isolated by dissection and cultured for 24 h in modified minimum essential medium in the presence of [³⁵S] methionine. De novo-synthesized proteins released into the culture medium were analyzed by two-dimensional PAGE and fluorography. Patterns of protein production by these isolated extraembryonic membranes remained relatively unchanged from days 23 to 45 of pregnancy. Electrophoretic profiles of proteins synthesized by allantois and amnion were identical but distinct from that produced by chorion. Yolk sac was the major source of serum-like proteins. An acidic (pI 5·3–6·3) 22 kDa protein, which consisted of four isoelectric variants, was produced by all extraembryonic membranes and demonstrated to immunoreact with antiserum produced against bovine placental retinol-binding protein (RBP). Limited N-terminal sequence analysis of one major isoform indicated that the protein had complete homology with bovine RBP over the first 15 amino acids. Immunoreactive RBP was localized in epithelial cells lining the chorion, allantois and amnion. In this study, we have characterized and compared protein production by isolated extraembryonic membranes through days 23 to 45 of pregnancy and identified the 22 kDa protein as caprine RBP of placental origin.

Journal of Endocrinology (1995) 146, 527–534

Pituitary gonadotropins mediate part of their effects on ovarian function via local hormones and growth factors produced by granulosa cells. Activins and inhibins are among these factors, and they have often opposite effects on various components of the reproductive system. The purpose of this study was to investigate the regulation of ovarian activin A secretion using cultured human ovarian granulosa-luteal cells as a model. The granulosa-luteal cells, obtained from women taking part in an in vitro fertilization program, were cultured and treated with FSH, LH, 8-bromo cAMP (8-BrcAMP, a protein kinase A activator) and 12-O-tetradecanoyl phorbol-13-acetate (TPA, a protein kinase C activator). Conditioned cell culture media were analyzed for activin A, inhibin A and progesterone concentrations with specific enzyme immunoassays. FSH and LH (1-100 IU/l) increased activin A secretion with 24 h of treatment (to 132% and 253% of control respectively; P<0.05 for both), but their effects were inhibitory in 48-h treatments (26% and 16% decreases respectively; P<0.05 for both). In the same experiments, FSH and LH increased inhibin A and progesterone secretion after both 24 and 48 h of treatment. 8-BrcAMP (0.1-100 muM) increased activin A in 24- and 48-h experiments (to 206% and 148% of control respectively; P<0.01 for both). Inhibin A and progesterone secretion were stimulated by 8-BrcAMP time- and dose-dependently. TPA increased activin A secretion dose-dependently (0.1-100 ng/ml) in both 24- and 48-h experiments. At 100 ng/ml concentration, it increased activin A up to 61-fold and inhibin A up to 16-fold of control in 24-h experiments. We conclude that gonadotropins regulate immunoreactive activin A secretion biphasically in cultured human granulosa-luteal cells: initial stimulation is followed by inhibition. In contrast, gonadotropins increase inhibin A and progesterone secretion continuously. Consequently, continuing gonadotropin stimulation leads to a decreasing activin:inhibin ratio, which may have a significant role in the local fine-tuning of ovarian steroidogenesis.

Our previous work showed that tumor necrosis factor (TNF)-α and FasL induce apoptosis of anterior pituitary cells. To further analyze the effect of these proapoptotic factors, we infected primary cultures from rat anterior pituitary, GH3 and AtT20 cells with first-generation adenoviral vectors encoding TNF-α, FasL or, as a control, β-galactosidase (β-Gal), under the control of the human cytomegalovirus promoter. Successful expression of the encoded transgenes was determined by immunocytochemistry. Although we observed basal expression of TNF-α and FasL in control cultures of anterior pituitary cells, fluorescence-activated cell sorting (FACS) cell cycle analysis showed that the overexpression of TNF-α or FasL increases the percentage of hypodiploid lactotropes and somatotropes. Nuclear morphology and TUNEL staining revealed that the cells undergo an apoptotic death process. We detected strong immunoreactivity for TNFR1 and Fas in the somatolactotrope cell line GH3. TNF-α, but not FasL, was expressed in control cultures of GH3 cells. The infection of GH3 cells with adenovirus encoding TNF-α or FasL increased the percentages of hypodiploid and TUNEL-positive cells. TNF-α or FasL immunoreactivity was not observed in the corticotrope cell line AtT20. However, adenovirus encoding TNF-α or FasL efficiently transduced these cells and increased the percentages of hypodiploid and TUNEL-positive cells. The expression of β-Gal was detected in all these cultures but did not affect cell viability. In conclusion, these results suggest that death signaling cascades triggered by TNF receptor 1 (TNFR1) and Fas are present in both normal and tumoral pituitary cells. Therefore, overexpression of proapoptotic factors could be a useful tool in the therapy of pituitary adenomas.

Thyroid dysfunction is classified into hyperthyroidism and congenital hypothyroidism (CH). Both hyperthyroidism and CH can cause heart lesions; however, the mechanisms involved remain unclear. The left ventricle was collected from eu-, hyper-, and hypothyroid rat. RNA was extracted and reverse-transcripted to cDNA. Real-time fluorescence quantitation-PCR was used to quantify the differential expression of thyroid hormone receptor (TR) subtype mRNA among eu-, hyper-, and hypothyroid rat myocardium. Here, we show that compared with the normal myocardium, TRα1 mRNA expression was upregulated by 51% (P<0.01), TRα2 mRNA expression was downregulated by 58% (P<0.01), and TRβ1 mRNA expression remained unchanged in hyperthyroid rat myocardium (P>0.05). TRα1, TRα2, and TRβ1 were expressed in normal and hypothyroid rat myocardium throughout the developmental process. In hypothyroid rats, myocardial TRα1 mRNA expression was generally downregulated and the expression peak appeared late. Myocardial TRα2 mRNA expression was generally upregulated and the expression peak appeared late. Myocardial TRβ1 mRNA expression was generally downregulated and changed similarly with the control group. In addition, the hypogenetic myocardium can be seen in the hypothyroid rat by pathology study. Taken together, the abnormal expression of TR subtype mRNA may have a close relationship with the pathogenesis of CH and hyperthyroidism heart disease.

Abstract

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030–1100 h on day 21 of pregnancy, increased significantly after 2200–2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.

Journal of Endocrinology (1996) 150, 479–486

Inhibins are gonadal glycoproteins with endocrine effects on pituitary FSH secretion and para/autocrine effects on ovarian and testicular function. The purpose of this study was to investigate the endocrine and para/autocrine regulation of inhibin A and inhibin B secretion in human ovarian granulosa-luteal cells. The cells were obtained from women undergoing in vitro fertilization, and the primary cultures were treated with FSH, LH, human chorionic gonadotropin (hCG), activin A, 8-bromo cyclic AMP (8-BrcAMP), staurosporine (a protein kinase C inhibitor) and an antagonist of IGF action (type-1 IGF receptor antibody alpha IR3). The secretion of inhibins was measured by ELISA assays capable of reliably distinguishing between inhibin A and B. FSH, LH, hCG and 8-BrcAMP increased inhibin A secretion on average up to 180% (P<0.01), 192% (P<0.05), 210% (P<0.01) and 243% (P<0.01) respectively of the control level, while their stimulatory effect on inhibin B secretion was less pronounced (up to 167%, P<0.01; 139%, P<0.05; 127%, P>0.05; 133%, P>0.05 of the controls respectively). alpha IR3 decreased inhibin A and B secretion down to 70% (P<0.01) and 50% (P<0.01) respectively of the control. Staurosporine decreased inhibin B secretion down to 49% (P<0.01) of the control; its effect on inhibin A secretion was not significant. Activin A increased inhibin B secretion up to fourfold of the control (P<0.05) while its effect on inhibin A secretion was insignificant. We conclude that gonadotropins via the protein kinase A signal transduction pathway are the main positive regulators of inhibin A and B secretion in human granulosa-luteal cells. The protein kinase C signal transduction pathway seems to be important especially for inhibin B secretion. Locally produced IGFs are probably important inducers of the production of both forms of inhibin in human ovaries while activins seem to upregulate inhibin B secretion.

Glucocorticoids (GCs) are routinely believed to work solely through genomic mechanisms. Recent evidence indicates that GCs can act at the membrane to exert rapid nongenomic effects on various tissues and cells. To ascertain whether nongenomic effects of GCs exist on the allergic asthma reaction, Hartley guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received inhaled budesonide (3 mg/ml suspended in Hydroxypropyl methylcellulose vehicle) for 5 minutes before ovalbumin challenge; Other animals received saline or blank vehicle as control. We measured the changes of lung resistance and dynamic lung compliance, the pulmonary function used to evaluate allergic asthma severity. Inhaled budesonide inhibited allergic reaction within 10 minutes, which would preclude genomic-mediated responses that normally takes several hours to occur. This study infers for the first time that rapid nongenomic effect of GCs exists on allergic asthma reaction, and provides a new way to investigate nongenomic mechanism of GCs. Further study would raise the possibility of new therapeutic strategies for allergic disease including asthma.