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

The progesterone (P₄) rise on proestrous afternoon is associated with dephosphorylation of tyrosine hydroxylase (TH) and reduced TH activity in the stalk-median eminence (SME), which contributes to the proestrous prolactin surge in rats. In the present study, we investigated the time course for P₄ effect on TH activity and phosphorylation state, as well as cAMP levels and protein phosphatase 2A (PP2A) activity and quantity, in the SME on proestrous morning and afternoon. P₄ (7.5 mg/kg, s.c.) treatment on proestrous afternoon decreased TH activity and TH phosphorylation state at Ser-31 and Ser-40 within 1 h, whereas morning administration of P₄ had no 1 h effect on TH. PP2A activity in the SME was enhanced after P₄ treatment for 1 h on proestrous afternoon without a change in PP2A catalytic subunit quantity, whereas P₄ treatment had no effect on PP2A activity or quantity on proestrous morning. cAMP levels in the SME were unchanged with 1 h P₄ treatment. At 5 h after P₄ treatment, TH activity and phosphorylation state declined coincident with an increase in plasma prolactin in both P₄-treated morning and afternoon groups. PP2A activity in the SME was unchanged in 5 h P₄-treated rat. Our data suggest that P₄ action on tuberoinfundibular dopaminergic (TIDA) neurons involves at least two components. A more rapid (1 h) P₄ effect engaged only on proestrous afternoon likely involves the activation of PP2A. The longer P₄ action on TIDA neurons is evident on both the morning and afternoon of proestrus and may involve a common, as yet unidentified, mechanism.

Abstract

We have analyzed the expression of the c-myc proto-oncogene in human adrenal glands in vivo and in primary cell cultures by Northern blot analysis. c-myc mRNA was consistently expressed in all human adrenals studied. Expression in adult adrenals was found to be approximately 50% of that in fetal adrenals, but much higher than that in adult liver and kidney. Adrenocorticotropin (ACTH) treatment increased c-myc mRNA accumulation dose- and time-dependently up to more than 5-fold (on average), with the maximal effect at 2 h. (Bu)₂cAMP and 12-O-tetradecanoyl phorbol 13-acetate (TPA) also induced c-myc gene expression. There was no synergistic effect between the ACTH, (Bu)₂cAMP and TPA treatments. The basal level of c-myc expression was reduced by the protein kinase inhibitors H-7 (1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride), staurosporine and HA1004 (N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). H-7 totally abolished ACTH-, TPA- and (Bu)₂cAMP-induced c-myc expression, while staurosporine inhibited the stimulatory effects of ACTH and TPA, and HA1004 weakly inhibited the effects of ACTH and (Bu)₂cAMP. Incubation with cycloheximide or 10% fetal calf serum increased c-myc mRNA levels 3- and 4-fold respectively. Our data show that the c-myc gene is expressed abundantly in normal human adrenals, and that this expression can be regulated by multiple factors in the primary cultures.

Journal of Endocrinology (1996) 148, 523–529

Abstract

Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ.

Journal of Endocrinology (1996) 151, 491–499

Abstract

Abundant c-myc gene expression in neoplasms has been often linked to poor prognosis. As c-myc mRNA is expressed and hormonally regulated in human adrenals, we examined the c-myc gene expression in adrenal tumors by RNA analysis and immunohistochemistry to find out the possible role of c-myc in adrenal neoplasms. The abundant expression of the c-myc gene in normal adrenals was localized to the zona fasciculata and zona reticularis, with much lower expression in the zona glomerulosa and adrenal medulla. In hormonally active adrenocortical carcinomas (n=6) and in virilizing adenomas (n=4), c-myc mRNA levels were approximately 10% of those in normal adrenals (n=11). In contrast, adrenal adenomas from patients with Cushing's (n=4) and Conn's (n=9) syndrome, non-functional adenomas (n=2), adrenocortical hyperplasias (bilateral, n=5; nodular, n=4), and non-functional adrenocortical carcinomas (n=3) expressed c-myc mRNA to the same extent as normal adrenals. The c-myc mRNA abundance in benign adrenal pheochromocytomas (n=19) was similar to that in normal adrenal medulla. However, in malignant adrenal pheochromocytomas (n=6), the average c-myc mRNA levels were approximately threefold that in benign adrenal pheochromocytomas. There was a good correlation between c-myc mRNA expression and immunohistochemical reactivity in both normal and pathological adrenal tissues. Southern blot analysis revealed no amplification or rearrangement of the c-myc gene in any of the adrenal tumors.

In conclusion, c-myc expression localized to zona fasciculata and reticularis in normal adrenals. Virilizing adenomas and hormonally active adrenocortical carcinomas expressed c-myc mRNA clearly less than the other adrenal neoplasms and normal adrenal tissue. On the other hand, malignant pheochromocytomas contained more c-myc mRNA than benign ones. Further studies are required to clarify the mechanisms and significance for the distinct expression pattern of the c-myc gene in different adrenal neoplasms.

Journal of Endocrinology (1997) 152, 175–181

Abstract

The steroidogenic acute regulatory protein (StAR) has recently been shown to be a factor necessary for cholesterol transport into adrenal and gonadal mitochondria, which is the regulated, rate-limiting step in steroidogenesis. We show here that StAR mRNA is highly expressed in normal adult adrenals (n=9), adrenocortical adenomas (n=16), adrenal hyperplasias (n=6), adrenocortical carcinomas (n=6) and adrenals adjacent to tumor tissues (n=9). There was a good correlation between the expression of StAR and the cholesterol side-chain cleavage enzyme/20,22-desmolase (P450 scc) mRNAs both in normal (r=0·93; P<0·01) and in tumor (r=0·97; P<0·001) tissues. No StAR mRNA was detected in Northern blots of liver, kidney, breast, parathyroid or phaeochromocytoma RNAs.

In cultured adrenocortical cells, adrenocorticotropin (ACTH), (Bu)₂cAMP, and cholera toxin increased StAR and P450 scc mRNA accumulation 6- to 18-fold, dose-and time-dependently. StAR (and P450 scc) mRNA increased relatively slowly in response to ACTH treatment, with the maximal increment at 24 h, while the mRNA of the early response gene c-fos peaked within 2 h. The protein kinase inhibitor H-7 inhibited basal and ACTH-induced StAR mRNA expression. Our results show that StAR mRNA is expressed at high levels in normal human adrenals and adrenocortical neoplasms. It is up-regulated in parallel with P450 scc by ACTH in adult adrenocortical cells, which suggests that ACTH is at least one of the key regulators of adrenal StAR expression.

Journal of Endocrinology (1996) 150, 43–50

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although hCG receptor transcripts were detected in human breast epithelial cells, raising the possibility of a direct mitogenic action, the hCG effect observed in this study may have been mediated via the ovary by increased secretion of ovarian steroids. In summary, using our in vivo nude mice system, the proliferation of normal human breast epithelial cells could be stimulated by host mouse pregnancy and by administration of hCG.

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.

Abstract

Human phaeochromocytomas abundantly express insulin-like growth factor-II (IGF-II), but its regulation and biological role in these neoplasms is not known at present. To clarify the regulation of IGF-II gene expression in phaeochromocytomas, we studied the effects of glucocorticoids, nerve growth factor (NGF), and protein kinase A and C regulators in primary cultures of human phaeochromocytoma cells. Cytoplasmic RNA was extracted and analysed by Northern and dot blotting with a ³²P-labelled cDNA probe for IGF-II. Dexamethasone treatment (500 ng/ml) for 3 and 7 days resulted in a 260% and 515% increase in the accumulation of IGF-II mRNA respectively. The stimulatory effect of dexamethasone was time-and dose-dependent. The increases in the 6·0 and 2·2 kb species of IGF-II mRNAs were the most apparent. Cortisol (1 μg/ml) increased the amount of IGF-II mRNA by threefold compared with the control. NGF (200 ng/ml), dibutyryl cyclic AMP (1 mm) and 12-O-tetradecanoyl phorbol-13-acetate (a protein kinase C activator; 100 ng/ml) had no significant effect on IGF-II mRNA levels. These data suggest that IGF-II gene expression in human phaeochromocytomas may be regulated by microenvironmental glucocorticoids.

Journal of Endocrinology (1994) 142, 29–35

Activin has previously been shown to act as a nerve cell survival factor and to have neurotrophic effects on neurons. However, the role of activin in regulating neurotransmitter expression in the central nervous system and the exact mechanisms involved in this process are poorly understood. In the present study, we report that activin A and basic fibroblast growth factor (bFGF) synergistically increased the protein level of tyrosine hydroxylase (TH), and also greatly increased the TH mRNA level, in both mouse E14 striatal primary cell cultures and the hippocampal neuronal cell line HT22. Activin A and bFGF cooperatively stimulated nuclear translocation of Smad3 and specifically activated ERK1/2, but not p38 or JNK. Interestingly, a specific inhibitor for MEK, U0126, efficiently blocked the induction of TH promoter activity by activin A and bFGF, indicating that activin A collaborated with bFGF signaling to induce the TH gene through selective activation of ERK-type MAP kinase in mouse striatal and HT22 cells. These data suggest that activin A may act in concert with bFGF for the development of TH-positive neurons.