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C Breton, D Di Scala-Guenot and HH Zingg

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.

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F. F. Bolander and M. E. Blackstone

ABSTRACT

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.

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P.F. Whitelaw, C.D. Smyth, C.M. Howles and S.G. Hillier

ABSTRACT

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.

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A. J. Beard, D. Savva, R. G. Glencross, B. J. McLeod and P. G. Knight

ABSTRACT

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.

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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.

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

The present study was designed to evaluate the regulation of nitric oxide (NO) synthesis in porcine oocytes during follicular development. Cumulus-oocyte complexes were obtained by aspirating the small follicles of immature porcine ovaries and cultured at 39 degrees C for 24-72 h with FSH in a serum-free medium. The oocyte-surrounding cumulus cells markedly proliferated and expressed LH receptor mRNA in response to FSH. The endothelial type of NO synthase (eNOS) (130 kDa) was detected in the oocyte, but not in the proliferated cumulus cells, by immunoblotting. The amount of oocyte eNOS did not significantly alter during culture, but measurement of nitrite and nitrate revealed FSH suppression of NO synthesis by approximately 50%. NO-releasing agents were added to the cultures to examine the effect of NO on the growth of cumulus cells. NO-releasing agents showed inhibitory effects on proliferation of the cumulus cells and expression of LH receptor mRNA. Thus, synthesis of eNOS-derived NO is suppressed in the porcine oocyte during development with no change in the enzyme amount, and it is suggested that it has an inhibitory function in the growth of cumulus cells.

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T. A. Yarney and M. R. Sairam

ABSTRACT

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.

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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.

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J. Brooks, W. J. Crow, J. R. McNeilly and A. S. McNeilly

ABSTRACT

The modulation of FSH secretion at the beginning and middle of the follicular phase of the cycle represents the key event in the growth and selection of the preovulatory follicle. However, the mechanisms that operate within the pituitary gland to control the increased release of FSH and its subsequent inhibition in vivo remain unclear. Treatment of ewes with bovine follicular fluid (bFF) during the luteal phase has been previously shown to suppress the plasma concentrations of FSH and, following cessation of treatment on day 11, a rebound release of FSH occurs on days 12 and 13. When luteal regression is induced on day 12, this hypersecretion of FSH results in an increase in follicle growth and ovulation rate. To investigate the mechanisms involved in the control of FSH secretion, ewes were treated with twice daily s.c. injections of 5 ml bFF on days 3–11 of the oestrous cycle and luteal regression was induced on day 12 with prostaglandin (PG). The treated ewes and their controls were then killed on day 11 (luteal), or 16 or 32h after PG and their pituitaries removed and halved. One half was analysed for gonadotrophin and gonadotrophin-releasing hormone (GnRH) receptor content. Total pituitary RNA was extracted from the other half and subjected to Northern analysis using probes for FSH-β, LH-β and common α subunit. Frequent blood samples were taken and assayed for gonadotrophins. FSH secretion was significantly (P<0.01) reduced during bFF treatment throughout the luteal phase and then significantly (P<0.01) increased after cessation of treatment, with maximum secretion being reached 18– 22h after PG, and then declining towards control values by 32h after PG. A similar pattern of LH secretion was seen after bFF treatment. Pituitary FSH content was significantly (P<0.05) reduced by bFF treatment at all stages of the cycle. No difference in the pituitary LH content was seen. The increase in GnRH receptor content after PG in the controls was delayed in the treated animals. Analysis of pituitary mRNA levels revealed that bFF treatment significantly (P<0.01) reduced FSH-β mRNA levels in the luteal phase. Increased levels of FSH-β, LH-β and α subunit mRNA were seen 16h after PG in the bFF-treated animals, at the time when FSH and LH secretion from the pituitary was near maximum. These results suggest that the rebound release of FSH after treatment with bFF (as a source of inhibin) is related to a rapid increase in FSH-β mRNA, supporting the concept that the rate of FSH release is directly related to the rate of synthesis.

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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).