Publications

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

Mol Cell Endocrinol. 2011 Aug 6;342(1-2):64-72

Authors: Kim SY, Zhu J, Woodruff TK

Abstract

The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production.

PMID: 21664424 [PubMed - indexed for MEDLINE]

Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system.

Gene expression in mouse ovarian follicle development in vivo versus an ex vivo alginate culture system.

Reproduction. 2011 Aug;142(2):309-18

Authors: Parrish EM, Siletz A, Xu M, Woodruff TK, Shea LD

Abstract

Ovarian follicle maturation results from a complex interplay of endocrine, paracrine, and direct cell-cell interactions. This study compared the dynamic expression of key developmental genes during folliculogenesis in vivo and during in vitro culture in a 3D alginate hydrogel system. Candidate gene expression profiles were measured within mouse two-layered secondary follicles, multi-layered secondary follicles, and cumulus-oocyte complexes (COCs). The expression of 20 genes involved in endocrine communication, growth signaling, and oocyte development was investigated by real-time PCR. Gene product levels were compared between i) follicles of similar stage and ii) COCs derived either in vivo or by in vitro culture. For follicles cultured for 4 days, the expression pattern and the expression level of 12 genes were the same in vivo and in vitro. Some endocrine (cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19a1) and inhibin βA subunit (Inhba)) and growth-related genes (bone morphogenetic protein 15 (Bmp15), kit ligand (Kitl), and transforming growth factor β receptor 2 (Tgfbr2)) were downregulated relative to in vivo follicles. For COCs obtained from cultured follicles, endocrine-related genes (inhibin α-subunit (Inha) and Inhba) had increased expression relative to in vivo counterparts, whereas growth-related genes (Bmp15, growth differentiation factor 9, and kit oncogene (Kit)) and zona pellucida genes were decreased. However, most of the oocyte-specific genes (e.g. factor in the germline α (Figla), jagged 1 (Jag1), and Nlrp5 (Mater)) were expressed in vitro at the same level and with the same pattern as in vivo-derived follicles. These studies establish the similarities and differences between in vivo and in vitro cultured follicles, guiding the creation of environments that maximize follicle development and oocyte quality.

PMID: 21610168 [PubMed - indexed for MEDLINE]

Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs.

Zinc sparks are triggered by fertilization and facilitate cell cycle resumption in mammalian eggs.

ACS Chem Biol. 2011 Jul 15;6(7):716-23

Authors: Kim AM, Bernhardt ML, Kong BY, Ahn RW, Vogt S, Woodruff TK, O'Halloran TV

Abstract

In last few hours of maturation, the mouse oocyte takes up over twenty billion zinc atoms and arrests after the first meiotic division, until fertilization or pharmacological intervention stimulates cell cycle progression toward a new embryo. Using chemical and physical probes, we show that fertilization of the mature, zinc-enriched egg triggers the ejection of zinc into the extracellular milieu in a series of coordinated events termed zinc sparks. These events immediately follow the well-established series of calcium oscillations within the activated egg and are evolutionarily conserved in several mammalian species, including rodents and nonhuman primates. Functionally, the zinc sparks mediate a decrease in intracellular zinc content that is necessary for continued cell cycle progression, as increasing zinc levels within the activated egg results in the reestablishment of cell cycle arrest at metaphase. The mammalian egg thus uses a zinc-dependent switch mechanism to toggle between metaphase arrest and resumption of the meiotic cell cycle at the initiation of embryonic development.

PMID: 21526836 [PubMed - indexed for MEDLINE]

In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment.

In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment.

Reproduction. 2011 Jul;142(1):113-22

Authors: Songsasen N, Woodruff TK, Wildt DE

Abstract

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E(2)) and progesterone (P(4)) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P(4) than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E(2) concentration remained unchanged over time (P>0.05) across FSH dosages. However, P(4) increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E(2) rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

PMID: 21502334 [PubMed - indexed for MEDLINE]

A method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system.

A method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system.

J Vis Exp. 2011;(49)

Authors: Shikanov A, Xu M, Woodruff TK, Shea LD

Abstract

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size. The degradable component may be particularly critical for clinical translation in order to support the greater than 10(6)-fold increase in volume that human follicles normally undergo in vivo .

PMID: 21445043 [PubMed - indexed for MEDLINE]

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates.

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and β-subunits as candidate substrates.

PLoS One. 2011;6(3):e17348

Authors: Antenos M, Lei L, Xu M, Malipatil A, Kiesewetter S, Woodruff TK

Abstract

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and β(B)-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.

PMID: 21408162 [PubMed - indexed for MEDLINE]

Structure of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and TGF-beta binding.

Structure of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and TGF-beta binding.

Proc Natl Acad Sci U S A. 2011 Mar 29;108(13):5232-6

Authors: Lin SJ, Hu Y, Zhu J, Woodruff TK, Jardetzky TS

Abstract

The zona pellucida (ZP) domain is a bipartite protein structural element comprised of ZP-N and ZP-C regions. Most notable for its ability to mediate protein polymerization, many ZP proteins polymerize and assemble into long fibrils that form specialized extracellular matrices. Other ZP proteins (namely, betaglycan and endoglin) do not polymerize but serve as important membrane coreceptors for ligands in the transforming growth factor-β (TGF-β) superfamily. Here, we present the 2.0-Å resolution crystal structure of the betaglycan ZP-C region in combination with a downstream region known as the external hydrophobic patch (EHP). Similar to the ZP-N region, the ZP-C region also adopts an immunoglobulin-like fold, despite sharing no sequence homology and possessing different disulfide linkages. The EHP region, which was previously thought to be external to the ZP region, is integral to the ZP-C domain and corresponds to the ZP-C G strand. Our structure also indicates that the critical maturation cleavage of ZP proteins, a process that activates nascent ZP proteins for polymerization, occurs within the immunoglobulin domain at the FG loop. Nonpolymerizing ZP proteins such as betaglycan and endoglin do not contain this cleavage site. Finally, our structure suggests that the AB loop and the convex surface pocket are regions important for TGF-β ligand binding.

PMID: 21402931 [PubMed - indexed for MEDLINE]

A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro.

A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro.

Reproduction. 2011 Jun;141(6):809-20

Authors: Tingen CM, Kiesewetter SE, Jozefik J, Thomas C, Tagler D, Shea L, Woodruff TK

Abstract

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.

PMID: 21389078 [PubMed - indexed for MEDLINE]

Altered gonadal expression of TGF-β superfamily signaling factors in environmental contaminant-exposed juvenile alligators.

Altered gonadal expression of TGF-β superfamily signaling factors in environmental contaminant-exposed juvenile alligators.

J Steroid Biochem Mol Biol. 2011 Oct;127(1-2):58-63

Authors: Moore BC, Milnes MR, Kohno S, Katsu Y, Iguchi T, Woodruff TK, Guillette LJ

Abstract

Environmental contaminant exposure can influence gonadal steroid signaling milieus; however, little research has investigated the vulnerability of non-steroidal signaling pathways in the gonads. Here we use American alligators (Alligator mississippiensis) hatched from field-collected eggs to analyze gonadal mRNA transcript levels of the activin-inhibin-follistatin gene expression network and growth differentiation factor 9. The eggs were collected from Lake Woodruff National Wildlife Refuge, a site with minimal anthropogenic influence, and Lake Apopka, a highly contaminated lake adjacent to a former EPA Superfund site. The hatchling alligators were raised for 13 months under controlled conditions, thus limiting differences to embryonic origins. Our data reveal sexually dimorphic mRNA expression in 13-month-old alligator gonads similar to patterns established in vertebrates with genetic sex determination. In addition, we observed a relationship between lake of origin and mRNA expression of activin/inhibin subunits α and βB, follistatin, and growth differentiation factor 9. Our study suggests that embryonic exposure to environmental contaminants can affect future non-steroidal signaling patterns in the gonads of a long-lived species.

PMID: 21251980 [PubMed - indexed for MEDLINE]

Hydrogel network design using multifunctional macromers to coordinate tissue maturation in ovarian follicle culture.

Hydrogel network design using multifunctional macromers to coordinate tissue maturation in ovarian follicle culture.

Biomaterials. 2011 Apr;32(10):2524-31

Authors: Shikanov A, Smith RM, Xu M, Woodruff TK, Shea LD

Abstract

Synthetic hydrogels with tunable properties are appealing for regenerative medicine. A critical limitation in hydrogel design at low solids concentration is the formation of defects, which increase gelation times and swelling, and reduce elasticity. Here, we report that trifunctional cross-linking peptides applied to 4-arm poly-(ethylene glycol) (PEG) hydrogels decreased swelling and gelation time relative to bi-functional crosslinkers. In contrast to bi-functional peptides, the third cross-linking site on the peptide created a branch point if an intramolecular cross-link formed, which prevented non-functional "dangling-ends" in the hydrogel network and enhanced the number of elastically active cross-links. The improved network formation enabled mouse ovarian follicle encapsulation and maturation in vitro. Hydrogels with bi-functional crosslinkers resulted in cellular dehydration, likely due to osmosis during the prolonged gelation. For trifunctional crosslinkers, the hydrogels supported a 17-fold volumetric expansion of the tissue during culture, with expansion dependent on the ability of the follicle to rearrange its microenvironment, which is controlled through the sensitivity of the cross-linking peptide to the proteolytic activity of plasmin. The improved network design enabled ovarian follicle culture in a completely synthetic system, and can advance fertility preservation technology for women facing premature infertility from anticancer therapies.

PMID: 21247629 [PubMed - indexed for MEDLINE]