Administration of long-term estradiol and progesterone followed by progesterone withdrawal does not alter the plasma oxytocin secretory response to cholecystokinin or the pituitary oxytocin content in ovariectomized rats.

The hormone oxytocin (OT) is important for several pre- and postpartum events, including uterine contractions at parturition, the induction of maternal behavior, and milk ejection during nursing. During late pregnancy, OT mRNA is increased in the paraventricular nucleus (PVN) due to high estrogen and declining progesterone levels. Administration of sequential estrogen and progesterone to, followed by withdrawal of progesterone from, an ovariectomized rat also increases OT mRNA. However, pituitary OT peptide is not affected. In the present experiment, we determined if this steroid exposure alters peripheral OT secretion during a provocative stimulus to OT release, such as cholecystokinin (CCK). Adult ovariectomized Sprague-Dawley rats were implanted on day 1 with either estrogen or empty silastic capsules, on day 3 with progesterone or empty capsules, and on day 14 progesterone or empty capsules were removed. Forty-eight hrs after removal of the progesterone capsules, plasma OT was measured before and after i.v. injection of 10 micrograms/kg of CCK. At the completion of the study, pituitary glands were removed and OT peptide was measured. No significant differences were found between the sham and hormone-treated animals either in their basal or CCK-stimulated plasma OT levels or their pituitary content of OT peptide. Although sequential exposure to estradiol and progesterone followed by withdrawal of progesterone has been shown previously to increase PVN OT mRNA, neither pituitary OT immunoreactivity nor basal and CCK-stimulated release of plasma OT is affected by this treatment. Although the mechanism of this steroid effect is not yet understood, our observations suggest a unique action of gonadal steroids upon PVN OT neurons.

Differential regulation of oxytocin and vasopressin messenger ribonucleic acid levels by gonadal steroids in postpartum rats.

We previously reported that sequential estradiol and progesterone exposure followed by progesterone withdrawal increases oxytocin (OT), but not arginine vasopressin (AVP), messenger ribonucleic acid (mRNA) in the hypothalamic paraventricular nucleus (PVN) of the rat. Substitution of testosterone for progesterone and subsequent testosterone withdrawal in the estrogen-primed rat increases PVN AVP mRNA levels. At the end of pregnancy (day 21), rats are exposed to high estrogen and declining progesterone and testosterone concentrations. Coincident with these changes in circulating gonadal steroid hormones in OT and AVP mRNAs. If progesterone levels are sustained at term, OT levels are attenuated and if testosterone is sustained, AVP mRNA levels are attenuated. Immediately postpartum, however, OT and AVP mRNA levels decline compared to term levels. To further determine the role of estrogen in the regulation of OT and AVP mRNAs, we performed two experiments. In the first experiment, we administered estrogen during the peripartum period to determine if estrogen supplementation prevents the relative attenuation of OT and AVP mRNAs that is seen after parturition. Day 18 pregnant rats were given estradiol-filled or empty capsules and sacrificed on day 2 lactation. By Northern analysis, significant differences in PVN AVP, but not OT, mRNA were found between the estrogen- and sham-treated lactational animals, P < 0.02. In the second experiment, we determined if sustaining estrogen after progesterone is removed in steroid-treated ovariectomized rats is essential for the increase in OT mRNA. Ovariectomized rats were given either empty capsules or sequential estradiol- and progesterone-filled capsules and both were sustained for 12 days. When progesterone-filled capsules were removed, estradiol-filled capsules were either removed or left in place, and the animals were sacrificed 48 h later. PVN OT mRNA was analyzed by Northern blot hybridization. OT mRNA was increased in both of the steroid-treated groups to the same degree, compared to sham-treated animals, P = 0.04. In summary, estrogen supplementation during early lactation prevents the attenuation of PVN AVP, but not OT, mRNA after parturition. In the estrogen-primed ovariectomized rat, it is not necessary to sustain estrogen to see the effects of progesterone withdrawal upon PVN OT mRNA.

Sequential exposure to estrogen and testosterone (T) and subsequent withdrawal of T increases the level of arginine vasopressin messenger ribonucleic acid in the hypothalamic paraventricular nucleus of the female rat.

The hypothalamic peptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated as mediators of socio-sexual behaviors in addition to their roles in osmolar homeostasis (AVP), milk ejection and uterine contractility (OT). Within 24 h of parturition, OT and AVP messenger ribonucleic acid (mRNA) levels increase in the hypothalamic paraventricular, and to a lesser degree, the supraoptic nucleus (PVN and SON) of the rat. We previously reported that the prepartum increase in OT mRNA is related to the spontaneous decline in progesterone levels prior to parturition. We also reported that increases in PVN and SON OT mRNA can be induced by exposing the ovariectomized rat to a steroid regimen that mimics the steroid milieu of pregnancy, namely sequential estrogen and progesterone and subsequent progesterone withdrawal. Levels of PVN and SON AVP mRNAs were not affected by progesterone withdrawal in late pregnant rats or the steroid regimen that increased OT mRNA in ovariectomized rats. These observations suggest that other factors, perhaps hormonal, may influence AVP mRNA levels. A decline in testosterone coincident with waning progesterone levels also occurs prepartum. Since peak levels of AVP mRNA prepartum coincide with the prepartum decline in testosterone, we questioned whether declining testosterone levels are important for the increase in AVP mRNA levels. To examine a possible role for testosterone in the increased level of AVP mRNA in late pregnancy, we sequentially administered estradiol and testosterone long-term (2 weeks) and removed testosterone 48 h prior to sacrifice. This steroid regimen mimics the estrogen and testosterone pattern of late pregnancy in rats. AVP, but not OT, mRNA levels increased significantly in the PVN of ovariectomized rats receiving this steroid regimen. We also found that implantation of late pregnant rats with testosterone capsules to prevent the spontaneous prepartum decline in testosterone, attenuates the increase in PVN AVP, but not OT, mRNA on day 21 of pregnancy. The data show that sequential estrogen and testosterone and testosterone withdrawal increase the level of PVN AVP mRNA in the female rat.

Positive elements in the laminin gamma 1 gene synergize to activate high level transcription during cellular differentiation.

Transcription of the murine laminin gamma 1 gene is activated during retinoic acid/cAMP induced differentiation of F9 embryonal carcinoma cells. Positive transcription control elements associated with two DNase I hypersensitive regions in the large first intron of the gene have been identified which confer a differentiation response on the laminin gamma 1 promoter. However, the kinetics of transcriptional activation suggest each DNA region interacts with transcription factors appearing at different times during differentiation. Synergy between the two regions in cis causes high level activation.

Effect of testosterone and its metabolites upon the level of vasopressin messenger ribonucleic acid in the hypothalamus of the hyperosmotically stimulated male rat.

We previously reported that gonadal steroids modify the expression of arginine vasopressin (AVP) in the hypothalamus of rats administered 2% sodium chloride solution for 5 days. Gonadectomy prevented, and testosterone (T) replacement restored, enhanced AVP mRNA levels in the supraoptic nucleus (SON) of male rats receiving this hyperosmotic challenge. The present study investigated the effects of the androgenic and estrogenic metabolites of T on hypothalamic AVP mRNA levels in response to chronic hyperosmolality. Gonadectomized male rats receiving 2% NaCl for 5 days and treated with T, dihydrotestosterone (DHT), or DHT+estradiol (E2), but not E2 alone or empty implants, had increased AVP mRNA levels compared to gonadectomized animals receiving tap water. Our results support a role for T and DHT-mediated effects upon the enhanced accumulation of AVP mRNA in the SON of male rats receiving a chronic hyperosmotic challenge.

Thyroidectomy does not alter hypothalamic oxytocin and vasopressin expression in chronically hypernatremic rats.

Sustained hyperosmolality increases the levels of hypothalamic oxytocin (OT) and arginine vasopressin (AVP) messenger ribonucleic acids (mRNAs). Gonadectomy is known to abolish this response (12,18). In this study we investigated whether thyroidectomy would alter OT and AVP mRNA levels in the hypothalamic paraventricular nucleus (PVN) of the hyperosmotically stimulated rat. Male Sprague-Dawley rats underwent thyroidectomy (hypothyroid) or sham thyroidectomy (euthyroid) at 7 weeks of age. Three weeks later hypothyroid and euthyroid animals were administered 2% NaCl (6-11 days) or tap water and sacrificed at the end of the experiment. Northern blot hybridization was used to assess size and levels of hypothalamic OT and AVP mRNAs. Hypothyroid rats had significantly lower levels of serum thyroxine (T4) than their euthyroid cohorts (P < 0.0001). Both the euthyroid and the hypothyroid animals receiving 2% NaCl developed hypernatremia and increased the levels and the size of OT and AVP mRNAs compared to their tap water cohorts. We conclude that in contrast to gonadectomy, thyroidectomy does not alter the level of OT and AVP mRNAs in the hypothalamus of chronically hypernatremic male rats.

Increased accumulation of oxytocin messenger ribonucleic acid in the hypothalamus of the female rat: induction by long term estradiol and progesterone administration and subsequent progesterone withdrawal.

To examine a possible role for gonadal steroid hormones in the enhanced accumulation of hypothalamic oxytocin (OT) messenger RNA (mRNA) and peptide in late pregnancy, we used an established model (22) in which sequential administration of estradiol (E2) and progesterone (P) SILASTIC capsules to ovariectomized rats is followed by removal of P. Long term and sustained E2 combined with abrupt P withdrawal mimics the gonadal steroid hormone pattern of late gestation in the rat (22). Using this paradigm, we demonstrate that OT mRNA is increased in the rat hypothalamus after long term P treatment, but only in the presence of E2 and only when P capsules are removed 48 h before killing. Furthermore, we show that P replacement in primiparous rats during late pregnancy blunts the increase in OT mRNA normally observed at the end of gestation. Our results support a role for E2 priming and P withdrawal in the enhanced accumulation of OT mRNA in the hypothalamus of the female rat.

Expression of the endothelin-1 and oxytocin genes in the hypothalamus of the pregnant rat.

Endothelin (ET)-1, a neuropeptide and possible neuromodulator, has been found in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) of the rat in the distribution of oxytocin (OT) neurons. Within the hypothalamus of the pregnant rat, we investigated the developmental expression of the ET-1 gene and the possibility of coordinate expression of the ET-1 and OT genes. Blots containing hypothalamic mRNAs from 4-, 14-, 18-, and 21-day-old pregnant rats were hybridized to a 32P-labeled probe specific to the rat ET-1 gene. Hypothalamic tissue contained an ET-1 transcript of approximately 2.3 kb size. ET-1 mRNA abundance increased significantly in the SON and PVN from early to late gestation (P = 0.005 and 0.05, respectively). Blots containing hypothalamic mRNA were rehybridized to a 32P-labeled probe specific to exon C of the rat OT gene. OT gene expression increased significantly within both the hypothalamic SON (p = 0.0009) and PVN (P = 0.003) as gestation advanced. The sizes of the hypothalamic ET-1 and OT transcript sizes remained unchanged throughout gestation. Hypothalamic ET-1 and OT transcripts display stage-specific increases during gestation. ET-1 may be a neuroendocrine regulator of pregnancy-related events in the rat, and may act alone or in concert with OT.

A survey of trace elements in fresh-water fish and rice along the Han River by neutron activation analysis.

For a case study of environmental pollution, radiochemical neutron activation analysis (RNAA) was applied to the crucian and rice collected along the Han River. The crucian was analyzed for three times in 1973, 1987, and 1990. Sixteen trace elements (Hg, Cd, As, Br, Cu, Na, K, Se, Cr, Hf, Rb, Fe, Zn, Co, La, and Cs) were determined by RNAA using distillation and diethyldithiocarbamate extraction methods. Contents of Na, K, Se, Hf, Fe, Zn, and Co were almost constant regardless of the sampling place and year. The contents of the other elements showed increasing trends down river, especially in the first investigation. At the lower part of the river, the contents showed decreasing trends with the time of sampling, especially during the first two investigations. These trends were typical for Hg and Cd. Rice was analyzed by the same method for 12 elements, and the results showed no regional trends, but have decreased after 1973.

Testosterone and hypothalamic vasopressin expression in the hyponatremic rat.

The level of serum osmolality is known to influence the expression of the arginine vasopressin (AVP) gene in the rat hypothalamus. Accumulation of AVP messenger ribonucleic acid (mRNA) is enhanced by sustained hypernatremia and diminished by sustained hyponatremia. We recently reported that gonadal steroid hormones modify the accumulation of hypothalamic AVP mRNA during sustained hypernatremia (12). Gonadectomized (gdx) hypernatremic male rats failed to increase AVP mRNA abundance, and testosterone (T) restored the response. Therefore, we questioned whether the decreased accumulation of AVP mRNA in experimentally-induced hyponatremic male rats is influenced, in part, by circulating concentrations of T, and determined if AVP mRNA accumulation would differ in the hypothalamus of gdx vs intact hyponatremic animals. Decreased accumulation of AVP mRNA was found in both intact and gdx hyponatremic animals compared to normonatremic animals. The level of AVP mRNA accumulation was not correlated with serum T concentrations. We conclude that diminished AVP in the hypothalamus of hyponatremic male rats occurs independent of circulating T concentrations.

Endothelin-1 gene expression in human pheochromocytoma.

We examined expression of human endothelin-1 (ET-1) in adrenal tissues removed from five patients with pheochromocytomas and two patients with aldosterone-producing adenomas. The pheochromocytomas contained a 2.3 kilobase ET-1 transcript, which was not found int he aldosteronomas. Constitutive expression of ET-1 varied in magnitude among the pheochromocytomas but was generally at a low level. Immunohistochemical staining of the pheochromocytomas with an antiserum to human ET-1 showed the presence of immunoreactive ET-1 as well. The presence of ET-1 messenger ribonucleic acid and ET-1 immunoreactivity in human pheochromocytomas suggests a possible paracrine role for this peptide in human chromaffin cells.

An environmental research on trace elements in freshwater fish by neutron activation analysis.

For a case study of environmental contamination, radiochemical activation analysis has been applied to the crucians collected in the Han River. Sixteen trace elements (Hg, Cd, As, Br, Cu, Na, K, Se, Cr, Hf, Rb, Fe, Zn, Co, La, and Cs) were separated into three groups using distillation and diethyldithiocarbamate extraction methods, and their contents were determined by a single comparator method. Compared with the values 15 years ago, the values for mercury and cadmium have been drastically decreased at the middle and lower part of the river, but no typical change is found in other elements.

The use of a chelating resin column for preconcentration of trace elements from sea-water in their determination by neutron-activation analysis.

A Chelex-100 resin column has been employed for the preliminary concentration of trace elements in water samples before their determination by neutron-activation analysis. The column, filled with a 1:1 mixture of the resin (50-100 mesh) and Pyrex glass powder of the same mesh-size, is shown to maintain a constant flow-rate and give reproducible results. By a combination of preconcentration and neutron-activation analysis it is possible to determine Ba, Ca, Cd, Ce, Co, Cr, Cu, Fe, La, Mg, Mn, Sc, U, V and Zn in sea-water and/or fresh water simultaneously at the parts per milliard level.