The differential, tissue-specific regulation of oxytocin (OT) binding sites allows the neurohypophysial nonapeptide OT to fulfill a dual role: to induce uterine contractions at parturition and to mediate milk ejection during lactation. Whereas uterine OT binding sites are up-regulated prior to parturition and are rapidly down-regulated thereafter, mammary gland OT binding sites gradually increase throughout gestation and remain up-regulated during the ensuing lactation period. Here, we structurally characterized OT receptor (OTR) mRNA in mammary gland and analyzed its expression during gestation and lactation and in response to steroid treatment. In mammary gland tissues, we found a 6.7 and a 5.4 kb OTR mRNA species, and both species were further analyzed by RACE (rapid amplification of cDNA ends). The 6.7 kb mRNA was found to be common to mammary gland and uterus and to extend 618 nucleotides beyond the published sequence of the rat OTR gene. The 5.4 kb mRNA species is unique to the mammary gland and terminates at a mammary gland-specific polyadenylation site that is not preceded by a classical polyadenylation signal. RT-PCR analysis did not provide any evidence for differences in the coding regions, suggesting that both uterine and mammary gland OTR mRNAs encode the same receptor protein. Furthermore, primer extension experiments showed that no differences exist in the specific transcriptional initiation sites of the OTR gene in the two tissues. During pregnancy, OTR mRNA per mammary gland increased approximately 150-fold and remained high during lactation, consistent with the previously identified regulation of OT binding sites and the role of OT during lactation. Whereas estrogen administration strongly induced the uterine OTR mRNA levels (>5-fold), mammary gland remained unaffected by steroid treatment. Moreover, tamoxifen had no effect on the mammary gland OTR mRNA level. In summary, our data demonstrate a differential control of OTR expression in uterus versus mammary gland and a mammary gland-specific OTR mRNA polyadenylation site. However, this differential control apparently does not involve the expression of different receptor genes nor the utilization of tissue-specific transcriptional initiation sites.
C Breton, D Di Scala-Guenot and HH Zingg
F. F. Bolander and M. E. Blackstone
The envelope protein, gp52, of the mouse mammary tumour virus (MMTV) binds to a cell-surface receptor as a first step in infection. A protein with characteristics of this receptor was measured on freshly isolated cells using, as ligand, 125I-labelled gp52 purified from C3H/HeN mice. The gp52-binding protein was found in all mouse tissues examined, but was present at highest concentrations in the mammary gland and spleen where it reached 4.2±0.3 (s.e.m.) pmol/mg protein; the dissociation constant was 30±7 pm. Binding to mammary epithelium could be displaced by either the RIII or 34I-R strains of MMTV, and binding was blocked by antibodies to gp52. Levels in the liver and adrenal glands were only 25% of those in the mammary gland, while the concentrations in the ovary and salivary gland were intermediate. Scatchard analyses of the binding data suggested that there was only a single set of high-affinity binding sites. During late pregnancy and lactation, receptor levels in mammary epithelium rose threefold, while those in the liver and salivary gland were unchanged. This induction would result in the mammary gland having 12 times the gp52-binding protein than other tissues and may result in the preferential reinfection of this tissue during lactation, with subsequent tumour formation.
GD Jahnke, MJ Miller, A Martinez, L Montuenga and F Cuttitta
Adrenomedullin (AM) is a recently identified amidated peptide produced by a variety of tissue types. We have investigated the involvement of AM and its receptor (AM-R) in developing mouse mammary glands and have examined what influence ovarian hormones have on AM and AM-R expression in this system. Tissues from ductal morphogenesis, virgin adult, pregnancy, and lactation stages were assessed for AM and AM-R by molecular, biochemical and immunohistochemical techniques. Results from these studies indicated that messenger RNA for AM and AM-R and immunoreactivity for AM were expressed in the luminal epithelium of small and large ducts and in terminal end buds. Immunoreactive AM was identified as a cytoplasm component of ductal cells, with some cells also having nuclear staining. Western blot analysis of mammary gland tissues yielded two molecular mass species (M(r) 14,000 and 18,500) of AM immunoreactivity in the mammary gland for the above developmental stages, consistent with processed intermediate and prohormone forms respectively. Ovariectomy alone or followed by hormonal treatments did not alter the expression pattern for these two proteins. By Western blot, the fully processed AM form (M(r) 6000) was identified in milk extracts from lactating glands. These data suggest a potential role for AM and its receptor in the maintenance of mammary gland homeostasis and suggests a potential role for AM in development of the newborn.
Elisabeth Sambroni, Antoine D Rolland, Jean-Jacques Lareyre and Florence Le Gac
The general rules established from mammalian species for the regulation of spermatogenesis by gonadotropins may not be fully relevant in fish. Particularly, Fsh is as potent as Lh to stimulate steroidogenesis and the Fsh receptor is expressed in Leydig cells. In seasonal breeders, Fsh is likely the major gonadotropin involved in spermatogenesis onset and Lh is required to support spermatogenesis progression and gamete release. However, the genes that relay the action of Fsh and Lh have been poorly investigated in fish. The present study was aimed at identifying gonadotropin-dependent genes expressed in the testis during fish puberty. We cultured pubertal trout testicular explants for 96 h, with or without gonadotropin, and analyzed transcriptome variations using microarrays. Fsh and Lh had similar effects on a large group of genes while other genes were preferentially regulated by one or the other gonadotropin. We showed that most of the responsive genes were expressed in somatic cells and exhibited relevant patterns during the seasonal reproductive cycle. Some genes preferentially modulated by Lh could be involved in testicular cell fate (pvrl1 and bty) or sperm maturation (ehmt2 and racgap1) and will deserve further examination. Besides Fsh's effects on the steroidogenic pathway, our study demonstrates that Fsh coordinates relevant stimulatory and inhibitory paracrine factors known to regulate early germ cell proliferation and differentiation. Some of these genes belong to major regulatory pathways including the Igf pathway (igf1b/igf3 and igfbp6), the Tgfb pathway (amh, inha, inhba, and fstl3), the Wnt pathway (wisp1), and pleiotrophin (mdka).
T. A. Yarney and M. R. Sairam
Differences in binding and structural properties of ovine testicular FSH and LH receptors were investigated. The ovine FSH receptor did not discriminate between FSH of different species, although equine FSH was more reactive. In the same tissue, however, the LH receptor showed marked preference for ovine and bovine LH, reacting very weakly with other preparations of pituitary LH. Human chorionic gonadotrophin also reacted partly with the ovine LH receptor at 25 °C. However, at 4 °C. the optimum temperature for binding of the LH receptor to its homologous hormone, the receptor displayed no recognition for chorionic gonadotrophin preparations. Affinity cross-linking studies with ovine testicular membrane suggested that the ovine FSH receptor has an M r of 70 000, which is very similar to that observed in the porcine ovary. The M r of the ovine LH receptor was estimated to be 150 000, which is different from those of other mammalian species, including those that have been cloned. The data suggest that the binding and structural properties of the ovine FSH receptor are similar to those of other mammalian FSH receptors, whereas the ovine LH receptor appears to differ from other mammalian LH receptors in having a different M r and in being more stringent in its requirement for pituitary LH.
A. J. Beard, D. Savva, R. G. Glencross, B. J. McLeod and P. G. Knight
To investigate the inhibin-induced suppression of FSH secretion by the anterior pituitary, chronically ovariectomized heifers (three per group) were treated for 56–58 h with either steroid-free bovine follicular fluid (bFF; 8 ml i.v. every 8 h) or 0·9% (w/v) NaCl (8 ml i.v. every 8 h). Blood was withdrawn at 8-h intervals for analysis of plasma concentrations of FSH and LH by radioimmunoassay. At the end of the treatment period, heifers were slaughtered and pituitary glands recovered for determination of gonadotrophin contents and levels of mRNA encoding FSH-β, LH-β, TSH-β and common α glycoprotein hormone subunits using [32P]cDNA probes in total RNA dot and Northern blot assays. Treatment with bFF markedly suppressed plasma FSH by 85% (P<0·001 compared with pretreatment period), but did not affect plasma LH concentrations. Plasma FSH and LH concentrations did not vary significantly in the saline-injected control heifers. The level of FSH-β subunit mRNA was reduced by 60% (P<0·001) in heifers treated with bFF, whereas no significant differences between control and bFF-treated heifers were observed in the levels of mRNA encoding LH-β, TSH-β or common α subunits. Treatments with bFF, however, did not affect pituitary content of either FSH or LH.
These results support the conclusion that inhibin exerts its selective suppressive effect on the secretion of FSH by the bovine pituitary, at least in part, by directly inhibiting expression of the gene encoding the FSH-β subunit.
JT Dickey and P Swanson
The effect of steroid hormone treatment on coho salmon (Oncorhynchus kisutch) was examined. The cDNAs for coho salmon FSH beta and LH beta subunits were cloned and sequenced using reverse transcriptase PCR. Northern blot analysis revealed that a single transcript of 1 kb for each of these subunits was present in the pituitaries of vitellogenic and spermiating coho salmon. RNase protection assays (RPAs) were developed to quantify FSH beta and LH beta subunit transcript levels. For the RPAs, antisense RNA probes and sense RNA standards were prepared from a region of the cDNAs which spanned the signal peptide and a portion of the mature protein. These RPAs were used to examine the effects of exogenous steroids including testosterone, estradiol-17beta (E2) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-P) in vivo, in coho salmon at three time points during the spring period of gonadal growth when plasma levels of FSH are increasing. Both testosterone and E2 increased steady state mRNA levels of LH beta, whereas E2 decreased steady state mRNA levels of FSH beta in one experiment. Thus, the RPAs were able to detect changes in steady state mRNA levels in response to exogenous steroid treatment. Plasma and pituitary levels of FSH and LH were also measured using RIA. Throughout the experimental series, FSH plasma levels decreased in response to exogenous testosterone and E2 administration, while 17alpha,20beta-P had no effect on FSH plasma levels. Plasma LH levels were not detected throughout the course of the experiment. Pituitary LH increased in response to testosterone and E2, while pituitary FSH levels did not change. 17alpha,20beta-P had no effect on pituitary FSH or LH content during the experiment. Thus, regulation of the gonadotropins in coho salmon occurs at both the transcriptional as well as the translational level. Testosterone and E2 appear to have negative feedback effects on FSH, but positive feedback on LH.
P.F. Whitelaw, C.D. Smyth, C.M. Howles and S.G. Hillier
Current understanding of the endocrine and paracrine regulation of follicular oestrogen synthesis predicts that aromatase cytochrome P450 (P450arom) mRNA is inducible by FSH in granulosa cells. LH receptor mRNA is constitutively expressed in thecal/interstital cells, and is also thought to be induced in granulosa cells in response to joint stimulation by FSH and oestrogen. This study provides direct evidence that FSH induces the ovarian P450arom gene selectively, perhaps exclusively, in the granulosa cells of Graafian follicles. FSH-induction of LH receptor mRNA occurs simultaneously but is independent of oestrogen synthesis per se.
D. A. Rodin, S. D. Abbot, G. Saade and R. N. Clayton
There are significant differences between rats and mice in the gonadal regulation of several aspects of gonadotroph function. To investigate whether these extend to the pretranslational regulation of FSH synthesis by gonadal steroids, we have measured FSH-β mRNA levels following gonadectomy and sex-steroid replacement and have related these to serum and pituitary FSH as a reflection of overall hormone synthesis.
In ovariectomized rats, FSH-β mRNA levels increased by 8 h, decreased, and then rose progressively over the next 28 days. A similar pattern of response was observed in orchidectomized rats. In mice, there were progressive increases in FSH-β mRNA levels in both males and females following gonadectomy, without evidence of the early peaks observed in rats. In both species, the change in FSH-β mRNA levels after gonadectomy was greater in females than in males. These changes in FSH-β mRNA following gonadectomy were paralleled by changes in the serum FSH concentration. In ovariectomized female rats and mice, pituitary FSH stores increased by 8 h and 3 days respectively, whereas in male rats, pituitary FSH content did not rise until 10 days after orchidectomy. The most striking species difference was the marked and prolonged reduction of pituitary FSH after orchidectomy of mice.
Treatment of rats and mice from the time of ovariectomy, with a dose of oestradiol that prevents increases in serum LH, only partially attenuated the rises in FSH-β mRNA and serum FSH and did not prevent the increase in pituitary FSH content. Treatment of intact or orchidectomized rats with testosterone suppressed FSH-β mRNA levels to 50% below intact control values without affecting pituitary FSH content. In mice, testosterone treatment for 10 days reduced the post-castration increase in FSH-β mRNA by only 26%, and prevented the fall in pituitary FSH content, although the increased serum concentration of FSH was unaffected.
In conclusion: (1) there is a good correlation between FSH-β mRNA levels and overall FSH biosynthesis in male and female rats and female mice, but this relationship is less obvious in male mice where pituitary FSH stores are not increased; (2) the inability of oestradiol to prevent completely the post-ovariectomy increase in FSH-β mRNA and FSH synthesis in female rats and mice indicates either that other gonadal products are necessary or that higher doses of oestradiol are required than for complete suppression of LH synthesis; (3) whilst the post-gonadectomy increases in FSH-β mRNA are larger in the female of both species, there are no major differences between rats and mice in the regulation of FSH-β gene expression by sex steroids.
Ilaria Cimmino, Francesco Oriente, Vittoria D’Esposito, Domenico Liguoro, Pasquale Liguoro, Maria Rosaria Ambrosio, Serena Cabaro, Francesco D’Andrea, Francesco Beguinot, Pietro Formisano and Rossella Valentino
The dramatic rise in obesity and metabolic syndrome can be related, at least in part, to environmental chemical factors such as Bisphenol-A (BPA). In this study, we aimed to understand the effects of low-dose Bisphenol-A on the human mature adipocytes and stromal vascular fraction (SVF) cells, obtained from subcutaneous mammary adipose tissue of overweight female patients, undergoing surgical mammary reduction. 24 and/or 48-h exposure to BPA 0.1 nM elicited significant increase of the inflammatory molecules interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemo-attractant protein 1α (MCP1α) and induced G protein-coupled estrogen receptor 30 (GPR30) levels more than two-fold both in mature adipocytes and SVF cells. These effects were similar to that obtained in the presence of GPR30-specific agonist G1 (100 nM) and were reverted by G15 (1 µM), a GPR30-selective antagonist. As a result of BPA-GPR30 signaling activation, fatty acid synthase (FAS) and leptin mRNA levels were significantly higher upon BPA exposure (P < 0.05) in mature adipocytes, with an opposite effect on adiponectin (ADIPOQ). In addition, an increase in SVF cell proliferation and ERK1/2 phosphorylation, was observed, compared to untreated cells. G15 reverted all of these effects. Interestingly, the action of BPA on SVF cell growth was mimicked by IL-8 treatment and was reverted by incubation with anti-IL8 antibodies. All these data suggest that BPA at 0.1 nM, a ten times lower concentration than environmental exposure, increases the expression of pro-inflammatory cytokines via GPR30 both in mature mammary adipocytes and in SVF cells with a possible involvement of IL-8.