Publications

An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo.

An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo.

Endocrinology. 2008 Jun;149(6):2970-9

Authors: Sinkevicius KW, Burdette JE, Woloszyn K, Hewitt SC, Hamilton K, Sugg SL, Temple KA, Wondisford FE, Korach KS, Woodruff TK, Greene GL

Abstract

Estrogen-nonresponsive estrogen receptor-alpha (ERalpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ERalpha, which significantly reduces ERalpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ERalpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ERalpha ligand-independent pathways were active. In addition, the synthetic ERalpha selective agonist propyl pyrazole triol (PPT) and ER agonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ERalpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ERalpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ERalpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ERbeta may modulate ERalpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ERalpha due to up-regulated uterine ERbeta levels.

PMID: 18339713 [PubMed - indexed for MEDLINE]

Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions.

Structural and biophysical coupling of heparin and activin binding to follistatin isoform functions.

J Biol Chem. 2007 May 25;282(21):15930-9

Authors: Lerch TF, Shimasaki S, Woodruff TK, Jardetzky TS

Abstract

Follistatin (FS) regulates transforming growth factor-beta superfamily ligands and is necessary for normal embryonic and ovarian follicle development. Follistatin is expressed as two splice variants (FS288 and FS315). Previous studies indicated differences in heparin binding between FS288 and FS315, potentially influencing the physiological functions and locations of these isoforms. We have determined the structure of the FS315-activin A complex and quantitatively compared heparin binding by the two isoforms. The FS315 complex structure shows that both isoforms inhibit activin similarly, but FS315 exhibits movements within follistatin domain 3 (FSD3) apparently linked to binding of the C-terminal extension. Surprisingly, the binding affinities of FS288 and FS315 for heparin are similar at lower ionic strengths with FS315 binding decreasing more sharply as a function of salt concentration. When bound to activin, FS315 binds heparin similarly to the FS288 isoform, consistent with the structure of the complex, in which the acidic residues of the C-terminal extension cannot interact with the heparin-binding site. Activin-induced binding of heparin is unique to the FS315 isoform and may stimulate clearance of FS315 complexes.

PMID: 17409095 [PubMed - indexed for MEDLINE]

Structural basis for a functional antagonist in the transforming growth factor beta superfamily.

Structural basis for a functional antagonist in the transforming growth factor beta superfamily.

J Biol Chem. 2005 Dec 2;280(48):40177-86

Authors: Cook RW, Thompson TB, Kurup SP, Jardetzky TS, Woodruff TK

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.

PMID: 16186117 [PubMed - indexed for MEDLINE]

Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.

Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.

Tissue Eng. 2003 Oct;9(5):1013-21

Authors: Pangas SA, Saudye H, Shea LD, Woodruff TK

The in vitro culture of immature ovarian follicles is used to examine the factors that regulate follicle development and may ultimately provide options for reproductive infertility. The objective of this study was to develop a three-dimensional in vitro culture system for the growth and development of individual granulosa cell-oocyte complexes. An alginate hydrogel was used to encapsulate immature mouse granulosa cell-oocyte complexes (GOCs) that were subsequently maintained in a serum-free in vitro culture. An overall incorporation efficiency of 50% was achieved. The complexes were assessed by transmission electron microscopy for changes in ultrastructure during in vitro growth. The architecture of the follicular complex was maintained during the encapsulation and the subsequent culture. The granulosa cells proliferated, and the oocytes also grew in volume and obtained the structural characteristics of mature oocytes including cortical granule formation, a well-developed zona pellucida with microvilli, normal mitochondria, and lattice-like structures in the cytoplasm. Oocytes retrieved and matured were able to resume meiosis, a necessary step for proper development. Thus, this system represents a new in vitro methodology for growth of individual granulosa cell-oocyte complexes.

PMID: 14633385 [PubMed - indexed for MEDLINE]

Inhibin binding protein (InhBP/p120), betaglycan, and the continuing search for the inhibin receptor.

Inhibin binding protein (InhBP/p120), betaglycan, and the continuing search for the inhibin receptor.

Mol Endocrinol. 2002 Feb;16(2):207-12

Authors: Bernard DJ, Chapman SC, Woodruff TK

Betaglycan (the TGFbeta type III receptor) and InhBP/p120 (a membrane-tethered proteoglycan) were recently identified as putative inhibin receptors. Here, we review the current state of knowledge regarding these two proteins with respect to their potential roles in inhibin biology. Importantly, neither protein appears to satisfy all of the criteria required for classification as a bona fide inhibin receptor. Betaglycan does not appear to be expressed in pituitary gonadotropes, the primary target of circulating inhibins, and InhBP/p120 does not bind inhibins in conventional receptor binding assays. While both proteins appear capable of promoting inhibin-mediated antagonism of activin signaling, neither appears to generate inhibin-specific intracellular signals. Recently, additional inhibin binding proteins were identified in inhibin target tissues, including pituitary and Leydig cells. Characterization of these proteins, coupled with ongoing investigations of betaglycan and InhBP/p120, will lead to a clearer understanding of mechanisms of inhibin action.

PMID: 11818494 [PubMed - indexed for MEDLINE]

Cellular localization of mRNA and protein: in situ hybridization histochemistry and in situ ligand binding.

Cellular localization of mRNA and protein: in situ hybridization histochemistry and in situ ligand binding.

Methods Cell Biol. 1998;57:333-51

Authors: Woodruff TK

Powerful methods for the detection of mRNA and proteins in cells and tissue sections have been developed since the mid-1980s. This chapter discusses the applications of in situ hybridization histochemistry and in situ ligand binding to cells in culture and tissue sections. In situ hybridization takes advantage of paired nucleotide interactions between a labeled probe (antisense strand) and the endogenous mRNA (sense strand). Following processing, the mRNA is localized through detection of the disintegration pattern of the radiolabeled probe. Protein-protein interaction is detected in a similar fashion. Proteins are radiolabeled and incubated with tissues that contain target-binding proteins or receptors. On processing, the interaction sites are localized through detection of the radiolabeled probe. The methods are rapid, sensitive, specific, and provide important information regarding the sites of mRNA synthesis, abundance of protein, and the ability of the ligand to interact with the receptor in restricted cellular populations. Application of these techniques to cells in culture allows in vitro manipulation of endogenous mRNA or protein with various hormones or growth factors and a method to detect the results.

PMID: 9648114 [PubMed - indexed for MEDLINE]

Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles.

Encapsulated three-dimensional culture supports development of nonhuman primate secondary follicles.

Biol Reprod. 2009 Sep;81(3):587-94

Authors: Xu M, West-Farrell ER, Stouffer RL, Shea LD, Woodruff TK, Zelinski MB

Abstract

In vitro ovarian follicle cultures may provide fertility-preserving options to women facing premature infertility due to cancer therapies. An encapsulated three-dimensional (3-D) culture system utilizing biomaterials to maintain cell-cell communication and support follicle development to produce a mature oocyte has been developed for the mouse. We tested whether this encapsulated 3-D system would also support development of nonhuman primate preantral follicles, for which in vitro growth has not been reported. Three questions were investigated: Does the cycle stage at which the follicles are isolated affect follicle development? Does the rigidity of the hydrogel influence follicle survival and growth? Do follicles require luteinizing hormone (LH), in addition to follicle-stimulating hormone (FSH), for steroidogenesis? Secondary follicles were isolated from adult rhesus monkeys, encapsulated within alginate hydrogels, and cultured individually for

PMID: 19474063 [PubMed - indexed for MEDLINE]

The role of the extracellular matrix in ovarian follicle development.

The role of the extracellular matrix in ovarian follicle development.

Reprod Sci. 2007 Dec;14(8 Suppl):6-10

Authors: Woodruff TK, Shea LD

Abstract

Regulation of ovarian follicle development depends on endocrine- and paracrine-acting hormones, the 3-dimensional architecture of the follicle, and the physical rigidity of the surrounding tissue. These 3 forces are integrated throughout the life cycle of the follicle to ensure appropriate hormone secretion, differentiation of the somatic cells, and maturation of the oocyte. The process of in-follicle maturation provides a new tool for understanding ovarian follicle development under the influence of these factors.

PMID: 18089604 [PubMed - indexed for MEDLINE]

Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary.

Neonatal exposure to estrogens suppresses activin expression and signaling in the mouse ovary.

Endocrinology. 2007 May;148(5):1968-76

Authors: Kipp JL, Kilen SM, Bristol-Gould S, Woodruff TK, Mayo KE

Abstract

In the ovary, the steroid hormone estrogen and the TGF-beta superfamily member activin are both produced by granulosa cells and they both have intraovarian functions. Emerging evidence has indicated an interaction of these two signaling pathways. Based on the fact that estrogen and activin can impact early follicle formation and development, we hypothesize that estrogen treatment may alter activin signaling in the neonatal ovary. Therefore, this study was designed to examine the effect of neonatal diethylstilbestrol (DES) and estradiol (E(2)) exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. CD-1 mouse pups were given daily injections of DES, E(2), or oil on postnatal d 1-5, and ovaries and sera were collected on d 19. Neonatal DES or E(2) exposure decreased the number of small antral follicles, induced multioocytic follicle formation, and decreased activin beta-subunit mRNA and protein levels. Consistent with local loss of beta-subunit expression, the phosphorylation of Smad 2, a marker of activin-dependent signaling, was decreased in the estrogen-treated ovaries. The decreased beta-subunit expression resulted in a decrease in serum inhibin levels, with a corresponding increase in FSH. Estrogen also suppressed activin subunit gene promoter activities, suggesting a direct transcriptional effect. Overall, this study demonstrates that activin subunits are targets of estrogen action in the early mouse ovary.

PMID: 17255206 [PubMed - indexed for MEDLINE]

A steroid-conjugated contrast agent for magnetic resonance imaging of cell signaling.

A steroid-conjugated contrast agent for magnetic resonance imaging of cell signaling.

J Am Chem Soc. 2005 Sep 28;127(38):13164-6

Authors: Lee J, Zylka MJ, Anderson DJ, Burdette JE, Woodruff TK, Meade TJ

We have synthesized the first steroid hormone-MR contrast agent conjugate designed to track the cell signaling process upon binding to a gene switch system. The derivative has a high relaxivity and when tested in vitro is active as a progesterone antagonist (RU-486). By combining a transcriptional system and a noninvasive imaging technology, such as MRI, it would be a powerful tool to research the cell signaling pathway in vivo.

PMID: 16173742 [PubMed - indexed for MEDLINE]