Tag Archives: Follistatin

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: DISCUSSION(3)


The question arises whether the effect of estradiol on FSHp gene expression, which was very dramatic (up to 70%), may be mediated by the relatively lesser decrease (<46%) in expression of activin. We hypothesize that a small reduction in free activin concentration may be sufficient to inactivate second messenger cascades and thereby greatly reduce synthesis and secretion of FSH as reported for GnRH receptor expression mediated by GnRH.

Ovine anterior pituitary cells locally synthesize follista-tin, and activin stimulates the production of follistatin, independently of inhibin. However, there appear to be important differences among species with respect to effects of activin on FSH secretion. The activin concentration necessary to stimulate FSH secretion in ovine gonadotropes is three times that required in rat gonadotropes (EC50 = 0.3 and 0.1 nM, respectively), and the corresponding increase in FSH secretion was 150% and 500% (respectively) of control values. Likewise, a more robust FSH response was obtained to lower concentrations of activin (EC50 = 0.18 nM) with cultured ovine pituitary cells from immature lambs. Thus, it appears that factors such as age, sex, and gonadal status of the donor animals may affect pituitary production of FSH via the activin/follistatin intrapituitary feedback loop.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: DISCUSSION(2)

The data reported herein confirm that during anestrus, estradiol can regulate secretion of gonadotropins by a direct action on the anterior pituitary gland in the absence of any hypothalamic factors. These effects of estradiol appear to be mediated, at least in part, by intrapituitary activin. Inhibition of ovine pituitary pB gene expression by estradiol is consistent with previous findings in rats in which the ovariectomy-induced increase in pB mRNA was prevented by treatment with estradiol. There is a potential estrogen response element (AGGTAAnnnTGACCT) in the 3′-untranslated region of the published sequence of the human activin pB gene that differs from the consensus estrogen response element (AGGTCAnnnTGACCT) by a single base. Thus, the potential for direct estrogenic regulation of activin production exists. Alternatively, it is possible that estradiol may alter the transcription of other pituitary factors that reduce expression of activin pB.

In addition to decreased secretion of FSH, we observed a specific reduction in expression of the FSHp subunit in response to treatment with estradiol in vitro. These results are in good agreement with previous observations made in vivo. During both breeding and nonbreeding seasons, estradiol reduced secretion of FSH and decreased expression of mRNA for the FSHp subunit.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: DISCUSSION(1)


Current concepts of regulation of gonadotrope function include the autocrine-paracrine regulation of FSH synthesis and secretion by activin and follistatin. Some reports have documented small but significant increases in mRNAs for activin pB and inhibin a after gonadectomy in rats. Alternatively, effects of activin action on expression of FSHp may be mediated via changes in production of follistatin. The amount of pituitary mRNA for follistatin increases during proestrus in the rat. This seems incongruous with the idea that follistatin sequesters activin and thereby reduces synthesis and secretion of FSH. Furthermore, pituitary content of follistatin and free activin are inversely related during the estrous cycle, and free activin peaks during the cycle and appears to be responsible for a large portion of the secondary surge of FSH in the rat.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: RESULTS

FSH and LH Release from Pituitary Cells after Estradiol Treatment

Estradiol reduced secretion of FSH in a dose-dependent manner (P < 0.001; Fig. 1A). In the presence of 0.01 and 1.0 nM estradiol, FSH concentrations were suppressed by 37% and 54%, respectively, of mean basal release in control cultures. In contrast, secretion of LH was significantly enhanced by estradiol (P < 0.001; Fig. 1B). In the presence of 0.01 and 1.0 nM estradiol, LH concentrations were increased to 237% and 267%, respectively, of mean basal release in control cultures.

FSH and LH Release from Pituitary Cells after Treatment with Anti-Activin

Anti-activin reduced mean secretion of FSH by 51% (P < 0.01) but did not influence secretion of LH (Fig. 2).

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: MATERIALS AND METHODS(3)


Slot Blot Analysis

To corroborate the RT-PCR results, steady state amounts of FSHp mRNA were also measured by slot blot analysis as previously described. Amounts of mRNA for activin pB and follistatin were insufficient for detection by this method. Polyadenylated (poly[A]+) RNA was prepared from pituitary cells immediately following incubation and its integrity was confirmed by Northern blot analysis. Quantification of mRNA encoding the FSHp subunit was performed as described previously. Samples of poly(A)+ RNA (600 ng) were applied to nylon membranes in duplicate using a slot blot apparatus and cross-linked by exposure to ultraviolet radiation. Membranes were hybridized to radiolabeled cDNA for bovine FSHp. Membranes were exposed to film for 3 days, after which they were stripped by washing in boiling 0.1% SDS and probed with radiolabeled p-actin cDNA (188 base pair-amplified PCR product; see above). This procedure allowed for normalization of unequal loading among RNA samples on the nylon membrane. Autoradiographs were analyzed using the NIH 1.52 image analysis program. Concentrations of mRNA are expressed as percent of control values.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: MATERIALS AND METHODS(2)


Estradiol (0.01 or 1.0 nM) was added to the cells for the final 24 h of culture. After treatment, medium was collected from all cultures and stored at -20°C until hormone assays were performed.

To immunoneutralize activin, monoclonal antibody to activin B (anti-activin, 25 ^g/ml, kindly supplied by Ralph Schwall, Genentech, Inc., San Francisco, CA) was added to individual wells (n = 5) of 24-well plates for the final 24 h of culture. At the end of treatment medium was collected and stored at – 20°C until hormone assays were performed.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: MATERIALS AND METHODS(1)


Animals and Experimental Design

Five sexually mature, western-range ewes were used during the anestrous season (June) in Colorado. Anestrous status was confirmed by the absence of ovarian activity at slaughter. Anterior pituitary glands were collected following anesthesia with sodium pentobarbital and exsanguina-tion. Tissues were removed and immediately placed in ice cold dissociation medium. All procedures involving animals were approved by the Colorado State University Animal Care and Use Committee and complied with National Institutes of Health (NIH) guidelines.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: INTRODUCTION(2)

Maintenance of normal reproductive function is dependent on the precise regulation of gonadotropin biosynthesis and secretion. Control of gene expression for the p subunit of both gonadotropins is regulated by circulating gonadal steroids as well as by hypothalamic factors, principally GnRH. In addition to GnRH and gonadal steroids, expression of mRNA for FSHp and secretion of FSH are under the control of the peptide hormones inhibin, activin, and follistatin. The heterodimeric inhibins, designated in-hibin A (apA) and inhibin B (apB) are produced primarily by gonads and act to reduce secretion of FSH. Acti-vins, which are homomeric or heteromeric dimers of the pA and pB chains, are produced in a wide variety of tissues and stimulate synthesis of FSH by direct action on gona-dotropes.

Activin Modulates Differential Effects of Estradiol on Synthesis and Secretion: INTRODUCTION(1)


Reproductive activity of sheep varies with season. Female sheep in temperate latitudes become anestrus during late winter or early spring and resume reproductive activity in late summer or early fall. Seasonal fluctuation in reproductive activity is most likely due to changes in photoperiod because similar changes in reproductive function are manifest in sheep exposed to differing artificial photoperiods in an otherwise constant environment. Photo-periodic cues modulate reproductive function in sheep through steroid-dependent and steroid-independent mechanisms.

Activin A and Follistatin Regulate Developmental Competence: DISCUSSION(4)

These results suggest that activin A does not generally support cell proliferation throughout the early development of bovine embryos but rather stimulates it for a limited period or at a limited developmental stage. Activin A may act on bovine embryos until the third cell cycle and may enhance subsequent development of embryos. The activation of embryonic transcription, which follows the transition from maternal to embryonic control of development, is known as zygotic gene activation (ZGA). In bovine embryos, this transition occurs at the 8-cell stage or earlier. The absence of appropriate ZGA leads to the failure of further embryo development beyond the early cleavage divisions. Therefore, the development-enhancing effect of activin A may be associated with ZGA. In addition, bovine embryos normally develop to the 8- to 16-cell stage and are transported from the oviduct into uterus at 3-4 days after ovulation. The developmental period during which addition of activin A to medium affected bovine embryo development in vitro in the present study (prior to the 9- to 16-cell stage at 90 h postinsemination) seems to coincide with the period during which bovine embryos are present in the oviduct in vivo. Since activin A is produced by oviduct epithelial cells in mice and cows, addition of activin A to embryo culture in vitro may reproduce the environment of the cleavage-stage embryos in the oviduct in vivo. proventil inhaler

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