Uterine Foxl2 regulates the adherence of the Trophectoderm cells to the endometrial epithelium.

BACKGROUND: Forkhead Transcription Factor L2 (FOXL2) is a member of the forkhead family with important roles in reproduction. Recent studies showed that FOXL2 is expressed in human and bovine endometrium and that its levels fluctuate during pregnancy. In this study, we aimed at evaluating the expression and function of FOXL2 in embryo implantation. METHODS: Mouse uteri at different days of pregnancy were isolated and analyzed for the expression and localization of FOXL2. A lentiviral strategy was further employed to either knockdown or overexpress FOXL2 in non-receptive human endometrial AN3-CA cells and in receptive Ishikawa cells, respectively. These genetically modified cells were compared to cells infected with a control lentivirus to determine the function of FOXL2 in trophectoderm cells adherence to Endometrial Epithelium was associated with the expression of genes known to be involved in acquisition of uterine receptivity. RESULTS: We report that FOXL2 is expressed in both, the luminal epithelium and the myometrium of the mouse uterus and that its expression declines prior to implantation. We found that endometrial cells expressing low FOXL2 levels, either endogenous or genetically manipulated, were associated with a higher attachment rate of mouse blastocysts or human Jeg3 spheroids and mouse blastocysts. In accordance, low-FOXL2 levels were associated with changes in the expression level of components of the Wnt/Fzd and apoptotic pathways, both of which are involved in uterine receptivity. Furthermore, FOXL2 expression was inversely correlated with G-protein signaling protein 2 (Rgs2) and cytokine expression. CONCLUSIONS: These results suggest that FOXL2 interferes with embryo attachment. Better understanding of the function of FOXL2 in the uterus would possibly suggest novel strategies for treatment of infertility attributed to repeated implantation failure.

FOXL2 in human endometrium: hyperexpressed in endometriosis.

The present study investigated expression and protein localization of FOXL2 messenger RNA (mRNA) in endometrium of healthy women and in patients with endometriosis during endometrial cycle. In endometriotic lesions, FOXL2 mRNA and protein were evaluated and a possible correlation with activin A mRNA expression changes was also studied. Endometrium was collected from healthy women (n = 52) and from women with endometriosis (n = 31) by hysteroscopy; endometriotic tissues were collected by laparoscopy (n = 38). FOXL2 gene expression analysis in endometrium of healthy women showed a significant expression and no significant changes in mRNA levels between proliferative and secretory phases; a similar pattern was observed in endometrium of patients with endometriosis. Immunohistochemical evaluation showed that FOXL2 protein localized in stromal and glandular cells and colocalized with SUMO-1. FOXL2 mRNA expression was 3-fold higher in endometriosis than in healthy endometrium (P < .01) and a positive correlation between FOXL2 and activin A mRNA was found (P < .05) in endometriosis. In conclusion, FOXL2 mRNA expression and its protein localization do not change during endometrial cycle in eutopic endometrium from healthy individuals or patients with endometriosis; the hyperexpression of FOXL2 in endometriotic lesions suggests an involvement of this transcriptional regulator, probably associated with activin A expression and related to the pathogenesis of endometriosis.

Cell-type specific modulation of pituitary cells by activin, inhibin and follistatin.

Activins are multifunctional proteins and members of the TGF-ß superfamily. Activins are expressed locally in most tissues and, analogous to the actions of other members of this large family of pleiotropic factors, play prominent roles in the regulation of diverse biological processes in both differentiated and embryonic stem cells. They have an essential role in maintaining tissue homeostasis in the adult and are known to contribute to the developmental programs in the embryo. Activins are further implicated in the growth and metastasis of tumor cells. Through distinct modes of action, inhibins and follistatins function as antagonists of activin and several other TGF-ß family members, including a subset of BMPs/GDFs, and modulate cellular responses and the signaling cascades downstream of these ligands. In the pituitary, the activin pathway is known to regulate key aspects of gonadotrope functions and also exert effects on other pituitary cell types. As in other tissues, activin is produced locally by pituitary cells and acts locally by exerting cell-type specific actions on gonadotropes. These local actions of activin on gonadotropes are modulated by the autocrine/paracrine actions of locally secreted follistatin and by the feedback actions of gonadal inhibin. Knowledge about the mechanism of activin, inhibin and follistatin actions is providing information about their importance for pituitary function as well as their contribution to the pathophysiology of pituitary adenomas. The aim of this review is to highlight recent findings and summarize the evidence that supports the important functions of activin, inhibin and follistatin in the pituitary.

The Local Control of the Pituitary by Activin Signaling and Modulation.

The pituitary gland plays a prominent role in the control of many physiological processes. This control is achieved through the actions and interactions of hormones and growth factors that are produced and secreted by the endocrine cell types and the non-endocrine constituents that collectively and functionally define this complex organ. The five endocrine cell types of the anterior lobe of the pituitary, somatotropes, lactotropes, corticotropes, thyrotropes and gonadotropes, are defined by their primary product, growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH) and follicle stimulating hormone (FSH)/luteinizing hormone (LH). They are further distinguishable by the presence of cell surface receptors that display high affinity and selectivity for specific hypothalamic hormones and couple to appropriate downstream signaling pathways involved in the control of cell type specific responses, including the release and/or synthesis of pituitary hormones. Central control of the pituitary via the hypothalamus is further fine-tuned by the positive or negative actions of peripheral feedback signals and of a variety of factors that originate from sources within the pituitary. The focus of this review is the latter category of intrinsic factors that exert local control. Special emphasis is given to the TGF-ß family of growth factors, in particular activin effects on the gonadotrope population, because a considerable body of evidence supports their contribution to the local modulation of the embryonic and postnatal pituitary as well as pituitary pathogenesis. A number of other substances, including members of the cytokine and FGF families, VEGF, IGF1, PACAP, Ghrelin, adenosine and nitric oxide have also been shown or implicated to function as autocrine/paracrine factors, though, definitive proof remains lacking in some cases. The ever-growing list of putative autocrine/paracrine factors of the pituitary nevertheless has highlighted the complexity of the local network and its impact on pituitary functions.

Impaired FSHbeta expression in the pituitaries of Foxl2 mutant animals.

Forkhead box L2 (FoxL2) is required for ovarian development and differentiation. FoxL2 is also expressed in the pituitary where it has been implicated in the development and regulation of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. Here, we show that FoxL2 is not required for the specification of gonadotropes; the pituitaries of Foxl2 mutant mice contain normal numbers of gonadotropes that express glycoprotein α subunit and LHß. Whereas the specification of gonadotropes and all other hormonal cell types is normal in the pituitaries of Foxl2 mutant animals, FSHß levels are severely impaired in both male and female animals, suggesting that FoxL2 is required for normal Fshb expression. The size of the pituitary is reduced in proportion to the smaller body size of Foxl2 mutants, with a concomitant increase in the pituitary cellular density. In primary pituitary cultures, activin induces FSH secretion and Fshb mRNA expression in cells from wild-type mice. In cells from Foxl2 mutant mice, however, FSH secretion is not detected, and activin is unable to drive Fshb expression, suggesting that the mechanism of activin-dependent activation of Fshb transcription is impaired. However, a small number of gonadotropes in the ventromedial region of the pituitaries from Foxl2 mutant mice maintain FSHß expression, suggesting that a FoxL2- and activin-independent mechanism can drive Fshb transcription. These data indicate that, in addition to its role in the ovary, FoxL2 function in the pituitary is required for normal expression of FSH.

FoxL2 and Smad3 coordinately regulate follistatin gene transcription.

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic alphaT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous alphaT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in alphaT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in alphaT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

A Smad-binding element in intron 1 participates in activin-dependent regulation of the follistatin gene.

Follistatins exert critical autocrine or paracrine control in many tissues by binding and bio-neutralizing activin and several other transforming growth factor-beta ligands. In the pituitary, activin acts locally to induce follistatin expression and thus modulate its own actions. This local feedback loop safeguards against excessive activin signaling and maintains the necessary balance of activin and follistatin tone. To better understand the mechanisms underlying the activation of follistatin by activin A, follistatin transcription was evaluated in gonadotrope-derived alphaT3-1 cells. Transient transfection experiments established that follistatin-luciferase plasmids that incorporate up to 2.86 kb of the upstream region of the rat follistatin gene are not induced by activin A in alphaT3-1 cells. On the other hand, plasmids that incorporate intron 1 are responsive to activin A and induced by a constitutively active form of ALK4. These experiments ultimately identified a conserved Smad-binding element (SBE1) in intron 1, between +1791 and +1795. In alphaT3-1 cells treated with activin A, SBE1 preferentially recruits Smad3, but not Smad2, and mediates Smad3-dependent activation of follistatin transcription. shRNA knockdown of endogenous Smad3 in these cells compromises SBE1-mediated transcription in response to activin A and interferes with its ability to positively regulate follistatin mRNA levels. The findings of the current work illustrate the critical role of intron 1 of the follistatin gene in mediating Smad-dependent effects of activin and regulating the expression level of this gene in some cell types, such as pituitary cells of gonadotrope lineage.

Pituitary actions of ligands of the TGF-beta family: activins and inhibins.

Activins, as members of the transforming growth factor-beta superfamily, control and orchestrate many physiological processes and are vital for the development, growth and functional integrity of most tissues, including the pituitary. Activins produced by pituitary cells work in conjunction with central, peripheral, and other local factors to influence the function of gonadotropes and maintain a normal reproductive axis. Follistatin, also produced by the pituitary, acts as a local buffer to bind activin and modulate its bioactivity. On the other hand, inhibins of gonadal origin provide an endocrine feedback signal to antagonize activin signaling in cells that express the inhibin co-receptor, betaglycan, such as gonadotropes. This review highlights the pituitary roles of activin and the mechanisms through which these actions are modulated by inhibin and follistatin.

Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin.

The precise regulation of the anterior pituitary is achieved by the cell-specific and combined actions of central, peripheral and local factors. Activins, inhibins, and follistatins were first discovered as gonadal factors with actions on FSH production from pituitary gonadotropes. With the realization that these factors are expressed in a wide array of tissues, including the pituitary, it became apparent that the functional importance of activins, inhibins, and follistatins extends beyond the reproductive axis and that they often exert their effects by autocrine/paracrine mechanisms. As members of the TGF-beta superfamily, activins and inhibins control and orchestrate many physiological processes and are vital for the development, the growth, and the functional integrity of most tissues, including the pituitary. Activins exert effects on multiple pituitary cell types but the best-characterized pituitary targets of the autocrine/paracrine function of activins are the gonadotropes. The autocrine/paracrine function of the activin-binding proteins, follistatins, constitutes an important local mechanism to modulate activin bioactivity while the restricted actions of gonadal inhibins to betaglycan-expressing gonadotropes provides a secondary mode of regulation of cell-specific actions of activins. The aim of this review is to highlight and evaluate experimental evidence that supports the roles of activins, inhibins, and follistatins as autocrine, paracrine, and/or endocrine modulators of the pituitary.

Regulation of follicle-stimulating hormone secretion by the interactions of activin-A, dexamethasone and testosterone in anterior pituitary cell cultures of male rats.

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHbeta mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin betaB mRNA levels while only DEX affected betaA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin betaB mRNA levels but had no effect on betaA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on betaB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.

Rat anterior pituitary folliculostellate cells are targets of interleukin-1beta and a major source of intrapituitary follistatin.

Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [(125)I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express alpha, betaA, and betaB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1beta (0.005-5 nM) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1-27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1beta, glucocorticoids, and other agents on the response of pituitary cells to activins.

Antagonism of activin by inhibin and inhibin receptors: a functional role for betaglycan.

Activin and inhibin research has provided important insight into reproductive physiology as well as many areas involving regulation of cell growth, differentiation and function. Progress in understanding the roles of these hormones in various cell and tissue types has been complimented by novel discoveries at the molecular level that have shed light on ligand/receptor interactions, signaling mechanisms and regulation. While the receptors and signaling pathway for activin are now well characterized, the molecular basis for inhibin action has remained relatively unclear. Here we summarize recent advances in understanding inhibin's mode of action focusing on our recent identification of betaglycan as an inhibin co-receptor capable of mediating inhibin action.

Effect of adenovirus-mediated overexpression of follistatin and extracellular domain of activin receptor type II on gonadotropin secretion in vitro and in vivo.

Activins are dimeric proteins that stimulate the synthesis and secretion of pituitary FSH by interacting with two classes of receptors, type I and type II, to initiate their intracellular signaling cascade. The extracellular domain of type II activin receptor (ActRII-ECD) contains all structural determinants sufficient for high affinity ligand binding. A soluble recombinant ActRII-ECD has been reported to attenuate FSH secretion from cultured rat anterior pituitary cells in response to exogenous activin A or endogenous activin B. Follistatin is a binding protein that acts as an extracellular factor to bind and inactivate activin. We constructed adenoviral vectors able to mediate expression of follistatin 288 (AdexCAFS288) and ActRII-ECD (AdexCAECD) and tested their biological activities both in vitro and in vivo. The data show that adenovirus-mediated overexpression of either ActRII-ECD or follistatin was able to attenuate FSH secretion by cultured rat anterior pituitary cells. However, AdexCAFS288 overexpression of follistatin was more effective than adenovirus-mediated overexpression of ActRII-ECD. In vivo, a single ip injection of AdexCAFS288 induced the expression of high levels of follistatin and resulted in the suppression of serum FSH levels in castrated male rats for up to 12 d postinjection. Infection with AdexCAFS288 had no effect on LH secretion in vitro or in vivo, demonstrating its selectivity. In conclusion, the results demonstrate the effectiveness of adenovirus-mediated overexpression of follistatin and ActRII-ECD to regulate FSH secretion and the potential of using this strategy as a tool to further define the critical role of activin/inhibin/follistatin circuitry in the modulation of the reproductive system.