Absence Of Hormone Treatment Research Articles

Artificial reproduction of wild and pond-cultured common tench (Tinca tinca) under controlled conditions

The common tench is an important thermophilic freshwater fish used for aquaculture diversification. This species is farmed in ponds as well as caught in the wild in many European and China. Artificial reproduction under controlled conditions is one of the most important part of modern aquaculture. In this study, two experiments were conducted in which the influence of hormonal agents (Ovopel and Ovaprim - experiment I) and the origin of the spawning stock (wild and pond-cultured - experiment II) on the success of reproduction in terms of ovulation rate (%), latency time (h), pseudo-gonadosomatic index (PGSI, %), hatching rate (%), abnormal larvae rate (%), and spawning efficiency coefficient (Se) were examined. While the wild fish could not reproduce in the absence of hormonal treatment with 0 % ovulation rate. In all tested hormonal treatment allowed to obtain high-quality eggs with hatching rates of over 83 %. Significant differences in latency time were observed among groups from different origins, such as differences in ovulation rate (79 % vs. 90 %, for pond-cultured and wild fish, respectively), PGSI (8.2 vs. 8.9), and hatching rate (63.8 % vs. 84.3 %), which showed significantly better quality in fish from the wild than those from pond culture. This means that when spawners of common tench of various origins are available, it is better to choose spawners from natural waters for artificial reproduction.

Endometriosis and menopause-management strategies based on clinical scenarios.

Endometriosis is largely considered a premenopausal disease with symptoms often improving during menopausal transition. However, 2%-4% of postmenopausal women are affected by endometriosis symptoms. At the same time, many peri- and postmenopausal women experience menopausal symptoms and inquire about treatment. Because of the estrogen-dependent nature of endometriosis, treatment with menopausal hormone therapy requires careful assessment of the patient but should nevertheless be considered. Recurrence of endometriosis symptoms and risk for malignant transformation are potential risks to weigh when prescribing menopausal hormonal therapy. Choice of treatment should be guided by the presence and severity of current endometriosis symptoms, nature of menopausal symptoms, risk assessment of potential contraindications for treatment in patient history, and preferences of the woman after an informative discussion. Recurrence of endometriosis symptoms in a postmenopausal patient should always prompt rigorous evaluation, both in the presence and absence of hormonal treatment. Many recommendations on the topic are based on expert opinion and new studies are urgently needed to obtain evidence for optimal patient care.

An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

OR20-04 Modeling Uterine Disorders Utilizing Adult Uterine Stem Cells

Endometriosis and uterine fibroids (leiomyomas) are benign gynecological disorders affecting 5-15% of women of reproductive age. They cause a wide range of symptoms including mild to severe pelvic pain and infertility. Due to a paucity of proper study models, hormonal and cellular mechanisms driving the pathology of endometriosis and fibroid development and growth remain unclear. Therefore, in the current study, we established 3D spheroid/organoid cultures from human uterine epithelial and Stro-1+/CD44+ myometrial stem cells and also from cells isolated from normal proliferative phase endometrium. Uterine organoid cultures were derived from endometrial epithelial and myometrial cells isolated from women who were not receiving exogenous hormones at the time of laparoscopy or hysterectomy. They were embedded in Matrigel, and grown in culture media. To determine whether spheroids/organoids were responsive to steroid hormones, the cultures were treated in presence or absence of estradiol (E2), progesterone (P4) or the combination (E2+P4) in serum free culture media. Time-dependent spheroid/organoid-growth curves and morphological analyses were used to define growth characteristics of endometrial and myometrial organoids. Subsequently, immunohistochemical colocalization of steroid hormone receptors (estrogen receptor alpha (ER-α) and progesterone receptor (PR-A\\B), alpha smooth muscle actin (α-SMA; myometrial cell marker), vimentin (stromal cell marker) and E-cadherin (endometrial epithelial cell marker) was assessed. Epithelial organoids expressed only E-cadherin in the absence of hormonal treatment. Myometrial organoids expressed α-SMA and vimentin. No expression of E-cadherin was observed in myometrial organoids. However, we observed the expression of ER-α and PR-A\\B when organoids were treated with E2+P4 in a time-dependent manner. Stro-1+/CD44+ myometrial stem cells differentiated into α-SMA and fibroblast/stromal cells and response to sex hormones. These findings suggest human uterine organoid cultures retained their characteristic cellular responses to E2+P4 and could be maintained long-term in ex vivo culture. Thus, the current 3D uterine organoid systems show high expansion capacity with retention of phenotypical and functional properties, which can be used for uterine pathophysiological studies, drug discoveries and drug repositioning.

SUN-438 Spontaneous Recovery of the Hypothalamic-Pituitary-Gonadal (HPG) Axis in a Female with Kallmann Syndrome

Background: Kallmann Syndrome (KS) is characterized by hypogonadotropic hypogonadism (HH) due to isolated GnRH deficiency (IGD) and anosmia. While reversal of KS has been described, predominantly in males, few cases have been described in females. We report a female with KS who demonstrated spontaneous recovery of her HPG axis. Case Report: A 34-year-old woman with a history of KS was referred to the NIH Clinical Center for evaluation. She was evaluated for absent thelarche and primary amenorrhea at age 18 when, due to her history of vigorous exercise and low BMI, she was assumed to have hypothalamic amenorrhea. At age 22, she was first noted to be anosmic by her endocrinologist who diagnosed her with KS. She underwent bilateral breast augmentation, and oral contraceptive pills (OCPs) induced regular menstrual bleeding. OCPs were self-discontinued after 1 year and amenorrhea recurred. By her late 20s, her level of exercise had decreased and her BMI normalized. She reported 3 episodes of spontaneous menstrual spotting in the six months preceding her visit, cyclical but irregular breast tenderness and emotional lability in the absence of hormonal treatment. Her medications included only fluoxetine. Physical examination revealed a BMI of 19.1 kg/m2, bilateral breast implants, a high-arched palate and bilateral fifth digit clinodactyly. Her laboratory evaluation revealed a serum LH of 8 U/L, FSH of 9 U/L and E2 of 19 pg/mL; all other anterior pituitary hormones were normal. Frequent blood sampling revealed a high-frequency pattern of LH pulsatility (24 pulses/24 hrs) with sleep augmentation and mean LH of 6.8 U/L. Formal smell testing confirmed anosmia. Her pelvic ultrasound revealed normal ovaries with follicles and normal uterus with a 4 mm endometrial stripe. A brain MRI was normal except for absent olfactory bulbs. Clinical testing of 14 IGD genes showed a novel heterozygous ANOS1 variant (c.298C>A p.Q100K) of unknown significance that was also present in her unaffected mother. A maternal half-uncle was reportedly anosmic but is now deceased. Discussion: This KS female exhibited spontaneous recovery of her HPG axis, which has been reported in very few cases. Her olfactory bulb aplasia is consistent with a congenital defect in GnRH neuronal development given their common ontogeny, suggesting that her HH and recovery were not simply due to changes in body weight nor exercise. Although females with heterozygous ANOS1 variants have been reported to demonstrate features of HH, the role of this novel ANOS1 variant remains unclear due lack of functional data and the presence of the same variant in her asymptomatic mother. Conclusions: Reversal of the reproductive phenotype in women with KS is rare with little known about their genetic abnormalities and long-term outcomes. Further study of such cases is warranted to gain insight into the mechanisms required for activation of the HPG axis.

The features of the expression of sex steroids hormone receptors, pro-inflammatory markers and cyclin-dependent kinase inhibitor protein p16ink4a in endometrium at external genital endometriosis

Background. A local inflammatory reaction in the endometrium caused by viral infection and associated with external genital endometriosis (EGE) can affect the implantation properties of endometrium and be one of the causes of infertility. The aim of the study was to investigate the expression of estrogen and progesterone receptors, pro-inflammatory markers and inhibitor of cyclin-dependent kinases p16ink4a in the endometrium of women with infertility associated with EGE. Materials and methods. The object of the study was the material taken from 133 patients aged 19-35 years. Inclusion criteria were the presence of external genital endometriosis confirmed by laparoscopy and the absence of hormone treatment for 6 months prior to this study. Endometrial biopsies were obtained on 19-24 day of the menstrual cycle. Histological and immunohistochemical studies of endometrial biopsies were performed using standard methods. The expression of estrogen (ER) and progesterone receptors (PgR), inflammatory markers (CD8+, CD20+, CD4+, CD138+) and inhibitor of cyclin-dependent kinase p16ink4a were examined by immunohistochemistry. The evaluation of the expression of studied markers was performed in the epithelium of the glands and endometrial stromal component separately, using semiquantitative method H-Score, as well as qualitative and quantitative methods in image analysis system “Morphology 5.0” (VideoTest, Russia). Statistical analysis was performed using statistical software (STATGRAPHICS v.6.0). Results. A significant decrease in the expression of ER and PgR in endometrial stromal component was noted on the background of the high incidence of chronic endometritis in patients with EGE. All patients of the main group had a positive expression of p16ink4a. Conclusions. The obtained results indicate a high incidence of disorders of eutopic endometrial secretory transformation in patients with infertility associated with external genital endometriosis. The high incidence of the virus associated with chronic endometritis and sensitivity violation of endometrial tissue to sex steroid hormones, such as progesterone, may be a major determinant of infertility of patients in this group.

Contemporary issues in primary amenorrhea: An experience from a Tertiary Care Center

Introduction:Amenorrhea is classified as primary if menstrual bleeding has never occurred in the absence of hormonal treatment. The clinical significance of a lack of regular menstrual cycles extends beyond reproductive concerns. Episodes of amenorrhea as short as 90 day may have implications for bone and cardiovascular health.Aims and Objective:To evaluate all patients presenting with primary amenorrhea in the Endocrinology OPD of Gauhati Medical College and Hospital.Materials and Methods:A total of 14 patients presenting to the Endocrinology OPD from March 2010 to May 2012 with a history of primary amenorrhea were included in the study. All patients were subjected to a detailed history, a thorough clinical examination, and relevant biochemical, hormonal, and radiological investigations.Result:In our study, the average age of presentation was 17.23 ± 4.2 years. Out of the 14 patients presenting with primary amenorrhea, 5 patients (35.71%) were found to have Turner′s syndrome, 2 (14.28%) had XX (pure) gonadal dysgenesis, 2 (14.28%) patients had XY gonadal dysgenesis (Swyer syndrome), 2 (14.28%) patients had Müllerian agenesis, 2 (14.28%) patients had hypothalamic amenorrhea, and 1 (7.14%) patient was found to have multiple pituitary hormone deficiency.Conclusion:In concordance with other studies, Turner′s syndrome, Müllerian agenesis, and gonadal dysgenesis are the commonest causes of primary amenorrhea in our study. However, in contrast to certain Western reports, primary amenorrhea rather than short stature remains the commonest cause for seeking medical evaluation in patients with Turner′s syndrome.

Growth of Mixed-Sex and Monosex Nile Tilapia in Different Culture Systems

Growth of 17α-methyltestosterone treated monosex Nile tilapia, Oreochromis niloticus was compared with hormone untreated mixed-sex fish in four different culture systems. The experiment had 2 X 4 factorial design: the first factor was presence or absence of hormone treatment (monosex and mixed-sex fish), the second factor was related to culture system (cistern, flow-through, pen and pond). Fish were cultured under similar feeding regime and stocking density for six months and different growth parameters such as body weight, length, daily weight gain (DWG), specific growth rate (SGR), proximate body composition and survival rate were analyzed. Monosex tilapia showed significantly higher (P-value 0.05) among mixed-sex and monosex fish and between different culture systems. Monosex tilapia in pond culture system showed the highest weight, DWG and protein content while mixed-sex tilapia in cistern culture system showed the lowest weight, DWG and protein content. Considering the results of this study, culture of monosex tilapia in earthen ponds might be considered the ideal method for tilapia culture in India.

Women sleep objectively better than men and the sleep of young women is more resilient to external stressors: effects of age and menopause.

The aims of this study were to: (i) assess gender differences of objective sleep patterns in a general population sample; (ii) evaluate the effects of menopause and hormone treatment (HT) on the sleep of the same cohort; and (iii) examine gender differences in sleep resilience towards external stressors. The participants were (i) 1324 subjects without sleep complaints, recruited from the general population of Central Pennsylvania that spent one night in the sleep laboratory and (ii) 66 young, healthy volunteers whose sleep was disturbed during night four by an external stressor, i.e. 24-h blood drawing (average of nights 2 and 3 versus night 4). Women compared with men in the general population sample had significantly higher percentage of sleep time, lower percentage of stage 1, and higher percentage of slow wave sleep. Also, menopause, in the absence of HT, was associated with prolonged sleep latency and decreased deep sleep. Finally, young, healthy women compared with men experienced less sleep disturbance because of blood draws as indicated by a significantly smaller change in per cent sleep time, and percentage of stage 1 sleep. These findings suggest that women without sleep complaints sleep objectively better across age than men and the sleep of young women is more resistant to external stressors. Also, gonadal hormones exert a beneficial effect on women's sleep. This gender dimorphism in sleep regulation may have been to protect women from the demands of infant and child care, and in part, might contribute to women's lower cardiovascular risks and greater longevity.

Novel human breast cancer cell lines IBH‐4, IBH‐6, and IBH‐7 growing in nude mice

Breast cancer is the most frequent cancer in women. However, in vivo hormone receptor positive and metastatic models are scarce. The aim of the present manuscript was to assess if the novel steroid receptor positive human cell lines IBH-4, IBH-6, and IBH-7 developed in our laboratory from primary infiltrant ductal carcinomas are good models to study in vivo human breast cancer. Cell lines or tumors were inoculated to nude mice in the presence or absence of hormone supplementation. Growth was analyzed by ANOVA followed by Tukey-Kramer's test. Steroid hormone expression was assessed by immunohistochemistry and Western blotting. The histology of the tumors was analyzed. IBH-4 and IBH-6 cells were inoculated to nude mice and 100% of the injected mice developed tumors in the presence or absence of hormone treatment, although tamoxifen inhibited growth. IBH-4 and IBH-6 cell lines in vivo gave rise to poorly differentiated carcinomas with areas of solid growth and sarcomatoid areas showing no morphological signs of epithelial differentiation. Distinct features of malignancy were observed. IBH-7 tumors in animals receiving estradiol were semi-differentiated adenocarcinomas. IBH-7 cells grew only in the presence of estradiol, but even with hormone addition, the tumor take was 20%. These tumors metastasized to the uterus and lung and vascular tumor emboli were evident. IBH-7 tumors were invasive and able to break through the peritoneum. As a conclusion, IBH-4 and IBH-6 are good models for studying tumor progression, whereas IBH-7 is a good model for tumor take, being metastatic and strictly estrogen-dependent.

Oestrogen‐mediated phosphorylation and stabilization of BRCA2 protein in breast

Disease-associated BRCA2 mutations typically result in protein truncations that delete the phosphorylation-regulated S3291 BRCA2 domain that interacts with Rad51. BRCA2 hereditary breast cancers are usually ER(+), differing from BRCA1 hereditary cancers, which are usually ER(-). We studied BRCA2 protein expression and S3291 phosphorylation in normal breast tissues and in sporadic breast cancers and observed that BRCA2 is expressed and phosphorylated in normal breast and 10 ER(+) breast cancers but not in 10 ER(-) breast cancers. In order to study this correlation between ER and BRCA2 expression, we studied ER(+) breast cancer cell lines. We found that a rapid increase in BRCA2 S3291 phosphorylation occurs following 17-beta-oestradiol (E2) treatment. This increase seen in BRCA2 total and phospho-S3291 protein levels was found to be unaffected with cycloheximide pre-treatment, but decreased following tamoxifen, ICI 182,780 or roscovitine treatment. This suggests a requirement for ER and cdk (cyclin-dependent kinase) in mediating the increased protein levels. MCF7 cell cycle distribution analysis following E2, in both the presence and absence of roscovitine (a cdk inhibitor), did not demonstrate any changes during an 8 h period, which further supports our hypothesis that mitogenic effects of E2 are not predominant at early time points. Studies with MG132 proteasome inhibitor and siRNA to skp2 support a model in which skp2-mediated proteasomal degradation of BRCA2 rapidly degrades BRCA2 protein in the absence of hormone treatment, which likely inhibits this pathway. E2 was shown to improve survival of MCF7 cells upon radiation treatment and roscovitine partially reversed this effect. We have demonstrated that BRCA2 protein is specifically expressed in ER(+) breast cancers and are investigating a pathway that may show a link between E2 action and BRCA2 protein function in breast cancer.

Changes in the levels of mRNA coding for gonadotropin Ibeta and IIbeta subunits during vitellogenesis in the common Japanese conger Conger myriaster

: The common Japanese conger Conger myriaster is known to mature up to the mid-vitellogenic stage without hormonal treatment, in contrast to the Japanese eel Anguilla japonica, which does not mature spontaneously under captive conditions. In order to obtain basic information on the process of gonadal development in anguilliformes under natural conditions, we have characterized cDNA of two distinct gonadotropin β−subunits (GTH-Iβ and -IIβ) in the common Japanese conger, and examined changes in their mRNA levels during vitellogenesis in the absence of hormonal treatment. Northern blot analysis revealed that the mRNA levels of GTH-Iβ were low at the perinucleolus stage, increased at the oil droplet stage, and peaked at the primary yolk globule stage. However, the levels decreased significantly at the secondary yolk globule stage. On the other hand, GTH-IIβ mRNA was first detected at the oil droplet stage, and showed a drastic increase with the progression of ovarian development. These results suggest that the two GTHs have distinct functional roles in oogenesis in anguilliformes where GTH-I is involved in the early stages of oogenesis, and GTH-II in the late stages of oogenesis.

Steroid hormone (hydrocortisone, oestradiol and testosterone) uptake, storage or induced synthesis in tetrahymena.

After cyclodextrin-coated 10(-6) m steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P(450)aromatase.

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed.

Follicle-stimulating hormone regulation on its receptor messenger ribonucleic acid levels in cultured rat granulosa cells

Our studies using immature rat granulosa cells cultured in serum-free medium on collagen-coated dishes indicated that FSH receptor mRNA levels do not change for at least 4 days of culture in the absence of hormone treatment. Addition of FSH (30 ng ml −1) led to a reduction of FSH receptor mRNA for a short time (6 h), followed by an increase in FSH receptor mRNA levels that reached maximum of around 200% of the initial level within 2–3 days after the addition of FSH. Following the addition of 10 nM PMA, FSH receptor mRNA levels were decreased to 50% of the pretreatment levels. During prolonged exposure to PMA, gradual recovery of the FSH receptor mRNA level was observed, and it was significantly higher than the control level at 48 h. The inactive phorbol ester 4 α-phorbol-12,13-didecanoate did not depress FSH receptor mRNA levels. Downregulation of the FSH receptor mRNA was detectable at a PMA concentration of 1 nM. The two predominant FSH receptor mRNA transcripts, ca. 5.5 and 2.4 kb, respectively, appeared to be equally affected by SH and PMA treatments. To examine the role of PKC mediation of the effect of FSH on FSH receptor mRNA levels, granulosa cells were treated with the PKC inhibitor, H-7, and FSH. Although, FSH receptor mRNA levels decreased to 50% of control in the cells treated with FSH alone, the addition of H-7 (0.1 nM) caused no decline in FSH receptor mRNA levels relative to the control in the cells treated with FSH. On the other hand, inhibition of FSH receptor mRNA by FSH was partially suppressed by the PKC-selective inhibitor bisindolylmaleimide. The mRNA turnover experiments showed that the half-life of FSH receptor transcripts was unaffected by PMA exposure.

Effects of Estradiol-17β and Progesterone on Mating Behavior in Female Lesser Bushbabies (Galago moholi) in Captivity

The effects of ovariectomy, estradiol-17β, and progesterone on female sexuality were investigated in a prosimian primate, the lesser bushbaby (Galago moholi). Each of eight ovariectomized, adult bushbabies was pair-tested with a male partner in the absence of hormone treatment, during estradiol-17β (E2) treatment (3.125 μg/0.1 ml), and during E2+ progesterone (0.125 mg/0.1 ml) treatment. Pretreatment females were sexually nonreceptive and nonattractive toward the males. In contrast, E2treatment elicited vaginal opening, partial or complete epithelial cell cornification, and female receptivity in all females and elicited the complete mating repertoire in seven pairs. Progesterone treatment opposed the facilitatory effects of E2by inhibiting female receptivity and epithelial cell cornification and inducing vaginal closure. Behaviors were quantified following changes in hormone treatment, vaginal physiology, and the presence or absence of intromission. Female attractiveness (male sexual arousal) was initiated during vaginal swelling and was maintained for the duration of vaginal opening, while female receptivity was manifested exclusively during the period of vaginal opening. Female proceptive behavior was mainly associated with the period of receptivity. This study provided evidence of a strict hormonal regulation of both behavioral and nonbehavioral aspects of female sexuality in a prosimian.

Tissue distribution, hormone regulation and evidence for a human homologue of the estrogen-inducible Xenopus laevis vitellogenin mRNA binding protein

17 beta-estradiol induces the synthesis of massive amounts of the hepatic mRNA encoding the Xenopus laevis egg yolk precursor protein, vitellogenin. Vitellogenin mRNA exhibits a half life of approx. 500 h when 17 beta-estradiol is present, and 16 h after removal of 17 beta-estradiol from the culture medium. We recently reported that Xenopus liver contains a protein, which is induced by 17 beta-estradiol and binds with a high degree of specificity to a binding site in a segment of the 3'-untranslated region (3'-UTR) of vitellogenin mRNA implicated in 17 beta-estradiol stabilization of vitellogenin mRNA. To determine if this mRNA binding protein was specific to this system, or if it was present elsewhere, and regulated by other steroids, we examined the tissue distribution and androgen regulation of this protein. Substantial amounts of the vitellogenin 3'-UTR binding protein were found in several Xenopus tissues including testis, ovary and muscle. In the absence of hormone treatment, lung and intestine contained minimal levels of the mRNA binding protein. Testosterone administration induced the vitellogenin 3'-UTR RNA binding protein in several tissues. Additionally, we found a homologous mRNA binding protein in MCF-7, human breast cancer cells. Although the MCF-7 cell protein was not induced by 17 beta-estradiol, the MCF-7 cell mRNA binding protein appears to be closely related to the Xenopus protein since: (i) the human and Xenopus proteins elicit gel shifted bands with the same electrophoretic mobility using the vitellogenin mRNA 3'-UTR binding site; (ii) The human and Xenopus proteins exhibit similar binding specificity for the vitellogenin 3'-UTR RNA binding site; and (iii) RNA from MCF-7 cells is at least as effective as RNA from control male Xenopus liver in blocking the binding of the Xenopus and human proteins to the vitellogenin mRNA 3'-UTR binding site. Its broad tissue distribution and regulation by both 17 beta-estradiol and testosterone suggests that this mRNA binding protein may play a significant role in steroid hormone regulation of mRNA metabolism in many vertebrate cells.

Effect of the ectomycorrhizal fungus Hebeloma hiemale on adventitious root formation in derooted Pinus halepensis shoot hypocotyls

The effect of the ectomycorrhizal fungus Hebeloma hiemale and of its culture filtrate on in vitro rooting of Pinus halepensis derooted shoot hypocotyls was studied in an attempt to determine if ectomycorrhizal fungi could enhance adventitious root formation in gymnosperms. Pinus halepensis hypocotyls did not root in the absence of hormonal treatment, whereas the rooting percentage was 87.3% in the presence of 5 μM indole-3-acetic acid (IAA). In the presence of tryptophan, which is a precursor of IAA, H. hiemale strongly enhanced rooting of hypocotyls cultivated in the absence of any hormonal treatment. In the presence of 0.1 mM tryptophan, the rooting percentage of the inoculated hypocotyls was 96.6%, whereas it was only 7.6% in the absence of the fungus. Hebeloma hiemale culture filtrate obtained in the absence of tryptophan did not contain IAA and did not stimulate rooting of the hypocotyls. In contrast, a culture filtrate obtained in the presence of tryptophan contained IAA; an ethyl acetate extract from this filtrate allowed 100% rooting. Different fractions were isolated by preparative thin-layer chromatography from the IAA-containing filtrate and studied for their effect on rooting. It was demonstrated that IAA was responsible for the rhizogenic activity of H. hiemale. These results suggest that ectomycorrhizal fungi which rapidly metabolize exogenously supplied tryptophan to IAA could be a suitable tool to enhance in vitro rooting of micropropagated gymnosperms. Key words: ectomycorrhizal fungus, indole-3-acetic acid, rooting, shoot hypocotyl, Hebeloma hiemale, Pinus halepensis.

Glucocorticoid regulation of the humoral immune system. I. In vivo effects of dexamethasone on IgA and IgG in serum and at mucosal surfaces.

The present studies were undertaken to determine whether glucocorticoids influence the levels of Ig in serum, saliva, and vaginal secretions. When measured by RIA, IgA levels in serum were elevated when increasing doses of dexamethasone, a potent synthetic glucocorticoid, were administered to intact- and adrenalectomized-ovariectomized rats. In contrast, IgA levels decreased in saliva and vaginal secretions over the same dose range. Time course studies indicated that the decline in salivary IgA, observed at 24 h after a single injection of dexamethasone, preceded a rise in serum IgA detected at 24 h after the second hormone treatment. Both responses were maximal at day 2 and did not change with further hormone exposure. After immunization and boosting with SRBC at two mucosal sites (intestinal Peyer's patch and uterine lumen), dexamethasone increased anti-SRBC IgA antibody levels in serum and reduced their presence in vaginal secretions. In contrast, anti-SRBC IgG-antibody levels in serum and vaginal secretions were reduced with hormone treatment. In the absence of hormone treatment, pooled sera from nonimmunized animals, when analyzed by HPLC, contained polymeric and dimeric IgA that was present in roughly equal proportion. In response to dexamethasone, polymeric IgA increased to a greater extent than did monomeric IgA. In summary, these studies demonstrate that dexamethasone alters the levels of IgA as well as specifically directed IgA and IgG antibodies in secretions and serum. Further, it suggests that glucocorticoid controlled IgA increases in serum and decreases in vaginal and salivary secretions may be due, in part, to a redistribution of polymeric IgA from mucosal surfaces to serum.

RNA complementary to ?-amylase mRNA in barley

Two experimental approaches demonstrate that different types of RNA complementary to α-amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B α-amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of α-amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious "hybridization" to rRNA, α-amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.