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Free access

Boris Thurisch, Shermi Y Liang, Nanette Sarioglu, Lutz Schomburg, Jörg Bungert and Christof Dame

Homozygous deficiency of the transcription factor Gata4 in mice causes lethality due to defects in ventral morphogenesis and heart tube formation. There is increasing evidence demonstrating that GATA4 function is also relevant for normal developed organ systems, including the heart and endocrinum. To analyze the implication of Gata4 beyond development, we generated transgenic mice expressing inducible small interfering RNA against Gata4. In longitudinal analysis, efficient suppression of Gata4 mRNA (down to 80% of wild-type levels) and protein expression in the heart was detected 38 days after induction of Gata4 short hairpin RNA. Decreased Gata4 expression was associated with reduction in the expression of known cardiac target genes, but the function of the heart remained unperturbed at 20–30% of normal Gata4 levels. Interestingly, Gata4 expression was almost abolished in the ovary and testis. This was accompanied in the testis by a significant reduction of GATA4 downstream target genes, such as the genes encoding Mullerian inhibiting substance (MIS) and steroidogenic acute regulatory (StAR) protein. By contrast, expression levels of Mis and Star were only slightly modified in the ovary, and concentrations of circulating FSH and LH were normal in female transgenic mice after induction of Gata4 short hairpin RNA. However, inhibition of Gata4 expression led to the formation of ovarian teratoma in 10% of females. Histology of the teratomas showed predominantly ectodermal and mesodermal structures. Our data demonstrate that Gata4 is critically involved in the function and integrity of the gonads in vivo.

Free access

K Takesue, MA Hattori, N Nishida, Y Kato and N Fujihara

The present study was designed to investigate nitric oxide (NO) synthesis and the expression of endothelial NO synthase (eNOS) gene in cultured porcine granulosa cells. Granulosa cells prepared from small follicles (1-4 mm diameter) were cultured in plastic dishes coated with fibronectin in chemically defined medium, and matured after 48 h of stimulation with FSH. The concentrations of nitrite and nitrate remained relatively constant until 42 h of stimulation, after which they increased significantly up to twofold at 48 h. NO synthesis was accompanied by an increase in cGMP. Gene expression for eNOS was studied by RT-PCR, and a PCR product of the expected size amplified. eNOS mRNA was expressed in the presence of FSH, but not in the absence of FSH. Although eNOS mRNA was not expressed in the initial period, it was expressed after 12 h of stimulation with FSH, and remained at a relatively constant level until 48 h. Expression of eNOS mRNA preceded expression of LH receptor mRNA, which showed a maximal level at 24 h of stimulation. These observations suggest that eNOS expression is not related to a rapid synthesis of NO in developing granulosa cells, and that the activation of NO synthesis is rigidly regulated in the initial period of development.

Free access

Bolander FF Jr

Prolactin (PRL) is capable of stimulating both calcium and nitric oxide (NO) accumulation in mammary epithelial cells within 15min. A calcium ionophore was also able to stimulate NO levels to an extent similar to that generated by PRL. Furthermore, maximal concentrations of PRL and the ionophore were not additive, suggesting that they were both using the same pathway, i.e. calcium. Finally, the depletion of intracellular calcium completely abrogated the effect of PRL on NO production. No other pathway known to affect NO synthase (NOS) influenced the action of PRL. Specifically, manipulations of protein phosphatase 2B, protein kinase B (PKB), protein kinase C (PKC), and arginine transport did not alter the activation of NOS by PRL. Therefore, the ability of PRL to stimulate NO production at 15min can be completely explained by its ability to elevate intracellular calcium.

Free access

Bolander FF Jr

Previous studies have shown that, in unstimulated mammary epithelial cells from virgin mice, prolactin receptors are retained intracellularly because of their incomplete N-glycosylation. Activation of the nitric oxide/cGMP pathway stimulates Nacetylglucosamine (NAG) transferase I activity, completion of terminal glycosylation, and redistribution of the receptors to the cell surface. In this study, it was shown that nitric oxide could stimulate the phosphorylation of NAG transferase I in intact cells and that the cGMP-dependent protein kinase (PKG) could directly phosphorylate the purified enzyme. Furthermore, this modification was associated with enhanced enzymatic activity. Conversely, this stimulation of activity was blocked in intact cells by coincubation with a PKG inhibitor and reversed in the immunoprecipitated enzyme by alkaline phosphatase treatment. Kinetic analysis revealed that this effect on enzyme activity was due to an increase in V(max) without any change in K(m). Therefore, it appears that the nitric oxide/cGMP pathway activates NAG transferase I via direct phosphorylation by PKG.

Free access

KA Hendry, KJ Simpson, KR Nicholas and CJ Wilde

The lactating tammar wallaby progressively alters the rate of secretion and composition of its milk to provide appropriate nutrition for the developing offspring, whose needs are signalled by changes in the pattern and efficiency of its sucking. Tammars are also capable of asynchronous concurrent lactation, when the mother provides a dilute milk for a newborn young permanently attached to the teat (phase 2A of lactation), and a concentrated milk from an adjacent mammary gland for a young-at-heel (phase 3). The relationship between suckling behaviour and milk secretion, and the ability of adjacent glands to function independently, suggests that milk secretion is controlled locally, within each mammary gland, by a mechanism sensitive to frequency and completeness of milk removal. To determine if tammar milk contains a factor able to control milk secretion, milk fractions have been screened in tissue and cell culture bioassays. A 6-30 kDa fraction of phase 3 whey was found to inhibit milk constituent synthesis and secretion in vitro, and inhibitory activity was associated with two discrete fractions obtained by anion exchange chromatography, which contained protein bands migrating anomalously at 66 kDa and 63 kDa in SDS-PAGE. These bands were recognised in Western blotting by antiserum raised against a bovine autocrine inhibitor of milk secretion. By the same criteria, milk secreted in phase 2B of tammar lactation, when milk secretion is low and suckling intermittent but less vigorous than phase 3, also contained a feedback inhibitor of milk secretion. The results indicate that, as in dairy animals, marsupial milk secretion is under local control through feedback inhibition by a milk protein, and raise the possibility that autocrine feedback may influence the transition from phases of low milk secretion (phase 2A, 2B) to a high rate in the final third phase of lactation.

Free access

Y Myal, B Iwasiow, H Cosby, A Yarmill, A Blanchard, D Tsuyuki, A Fresnoza, ML Duckworth and RP Shiu

The human prolactin-inducible protein/gross cystic disease fluid protein-15 (PIP/GCDFP-15) gene is expressed in more than 90% of human breast cancer biopsies but not in the normal mammary gland. However, it is expressed in several normal human apocrine glands such as the lacrimal and salivary glands. In human breast cancer cell lines, the gene is regulated by a number of hormones including androgen and prolactin. It is not known whether gene expression in normal tissues is under similar hormonal control. To understand the mechanisms by which hormone- and tissue-specific expression of the human PIP/GCDFP-15 gene are regulated in vivo, we generated transgenic mice using a 13.7 kb genomic DNA fragment containing the entire 7 kb human gene, together with 2.9 kilobases of 5' and 3.8 kilobases of 3' flanking sequences. The human PIP/GCDFP-15 transgene was found to be expressed in both the lacrimal and salivary glands but was not expressed in the mammary glands of transgenic mice. This tissue-specific pattern of the transgene expression in the mouse was very similar to that of the endogenous human PIP/GCDFP-15 gene, and to the endogenous mouse,gene. In the mouse salivary glands, the transgene expression was highest in the parotid, considerably less in the submaxillary (submandibular) and absent in the sublingual glands. In the mouse lacrimal gland, as in the human breast cancer cell lines, the human PIP/GCDFP-15 transgene was also up-regulated by androgen. These studies demonstrate that the human gene with its 6.3 kb flanking sequences is able to confer gene expression in vivo in a tissue-specific and hormone-responsive manner.

Free access

G Pelletier, V Luu-The, M El-Alfy, S Li and F Labrie

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.

Free access

Sunan Li, Juxiong Liu, Qingkang Lv, Chuan Zhang, Shiyao Xu, Dongxue Yang, Bingxu Huang, Yalong Zeng, Yingjie Gao and Wei Wang

Abstract

The ghrelin peptides were found to circulate in two major forms: acylated ghrelin (AG) and unacylated ghrelin (UAG). Previous studies showed that AG regulates β-casein (CSN2) expression in mammary epithelial cells. However, little is known about the mechanisms by which AG regulates CSN2 gene and protein expression. Evidence suggests that UAG has biological activity through GHSR1a-independent mechanisms. Here, we investigated the possible GHSR1a-mediated effect of UAG on the expression of CSN2 in primary bovine mammary epithelial cells (pbMECs) isolated from lactating cow. We found that both AG and UAG increase the expression of CSN2 in a dose-dependent manner in pbMECs in comparison with the control group. Increased expression of CSN2 was blocked by [D-Lys3]-GHRP-6 (an antagonist of the GHSR1a) and NF449 (a Gs-α subunit inhibitor) in pbMECs. In addition, both AG and UAG activated AKT/protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, whereas [D-Lys3]-GHRP-6 and NF449 inhibited the phosphorylation of AKT and ERK1/2 in pbMECs respectively. Blockade of ERK1/2 and AKT signaling pathways prevented the expression of CSN2 induced by AG or UAG. Finally, we found that both AG and UAG cause cell proliferation through identical signaling pathways. Taken together, these results demonstrate that both AG and UAG act on ERK1/2 and AKT signaling pathways to facilitate the expression of CSN2 in a GHSR1a-dependent manner.

Restricted access

B. Quérat, M. Moumni, M. Jutisz, Y.A. Fontaine and R. Counis

ABSTRACT

Oestradiol treatment enhances type-II gonadotrophin (GTH-II) synthesis in the European eel (Anguilla anguilla) at the silver stage. As a first step in studying the molecular mechanisms involved in this stimulation, we cloned and characterized the cDNA encoding the β subunit of eel GTH-II.

A cDNA library was constituted in λgt10 from oestradiol-treated eels. It was screened using an oligodeoxyribonucleotide mixed probe designed to be complementary to a highly conserved region of cDNAs from several LH-related β subunits. Several clones were obtained and four were subcloned in pUC13 and sequenced. The longest clones comprised a 420 bp coding sequence, plus 5′ and 3′ untranslated regions of 36 and 172 bp respectively. Comparison with GTH-II from other teleost fish permitted the localization of the putative cleavage site of a 24 amino acid signal peptide. The resulting 116 amino acid apopeptide had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 70–80% with GTH–II from other fish, 45% with LH from mammals and birds, 38% with mammalian FSH and only 35% with fish GTH-I. Preliminary results indicated a strong positive effect of oestradiol treatment on the level of the putative GTH-II β-subunit mRNA. This supports our proposal that the European eel provides a suitable model for studying the positive regulation of gonadotrophin synthesis by gonadal steroids.

Restricted access

S J B Aylwin and J M Burrin

Introduction

The glycoprotein hormone α-subunit (αSU) is the common element in a family of heterodimeric glycoprotein hormones which includes luteinising hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH) and human chorionic gonadotrophin (Pierce & Parsons 1981). The gene encoding the αSU exists as a single copy gene expressed in pituitary thyrotrophs and gonadotrophs of all mammals studied, but placental expression has only been found in primates and horses (Fiddes & Goodman 1981, Fiddes & Talmadge 1984, Fox & Solter 1988, Fenstermaker et al. 1990). During ontogeny, unique pituitary cell types differentiate in a sequential pattern and begin to synthesise and secrete specific hormones (Simmons et al. 1990, Borrelli 1994). The anterior pituitary develops from an invagination of the pharyngeal epithelium to form Rathke's pouch, and in situ hybridisation studies in the mouse have detected αSU mRNA in this region as early as embryonic day 11·5 (Simmons et al. 1990). At