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Many women suffer from either failed fertilisation or their embryos arrest early during development. Autologous mitochondrial supplementation has been proposed as an assisted reproductive technology to overcome these problems. However, its safety remains to be tested in an animal model to determine if there are transgenerational effects. We have supplemented oocytes with autologous populations of mitochondria to generate founders. We mated the female founders and their offspring to produce three generations. We assessed litter size, the ovarian reserve, and weight gain and conducted a full histopathological analysis from each of the three generations. Across the generations, we observed significant increases in litter size and in the number of primordial follicles in the ovary matched by changes in global gene expression patterns for these early-stage oocytes. However, full histopathological analysis revealed that cardiac structure was compromised in first and second generation offspring, which could seriously affect the health of the offspring. Furthermore, the offspring were prone to increased weight gain during early life. Mitochondrial supplementation appears to perturb the regulation of the chromosomal genome resulting in transgenerational phenotypic gains and losses. These data highlight the need for caution when using autologous mitochondrial supplementation to treat female factor infertility.

Mature TGF-β proteins are used in vivo to promote bone growth, combat obesity, reverse fibrosis and pulmonary arterial hypertension, and as potential rejuvenation factors. However, the serum half-life of this family of growth factors is short (∼5 min), limiting their therapeutic potential. Because TGF-β proteins are normally secreted from cells with their prodomains attached, we considered whether these molecules could extend the in vivo half-life and activity of their respective growth factors. Using activin A as a model ligand, we initially modified the cleavage site between the pro- and mature domains to ensure complete processing of the activin A precursor. Co-immunoprecipitation studies confirmed mature activin A is secreted from cells in a non-covalent complex with its prodomain, however, the affinity of this interaction is not sufficient to suppress activin A in vitro biological activity. The plasma clearance profiles of purified pro- and mature activin A were determined over a 4 h period in adult male rats. Both activin forms demonstrated a two-phase decay, with the half-life of pro-activin A (t1/2 fast = 12.5 min, slow = 31.0 min) being greater than that of mature activin A (t1/2 fast = 5.5 min, slow = 20.3 min). Both pro- and mature activin A induced significant increases in serum follicle stimulating hormone levels after 4 h, but no differences were observed in the relative in vivo bioactivities of the two activin isoforms. Increased serum half-life of activin A in the presence of its prodomain identifies a new means to increase the therapeutic effectiveness of TGF-β proteins.

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70-80 µm); yet, survival was low and smaller follicles (

Hydrogel-encapsulating culture systems for ovarian follicles support the in vitro growth of secondary follicles from various species including mouse, non-primate human, and human; however, the growth of early stage follicles (primary and primordial) has been limited. While encapsulation maintains the structure of early stage follicles, feeder cell populations, such as mouse embryonic fibroblasts (MEFs), are required to stimulate growth and development. Hence, in this report, we investigated feeder-free culture environments for early stage follicle development. Mouse ovarian follicles were encapsulated within alginate hydrogels and cultured in various growth medium formulations. Initial studies employed embryonic stem cell medium formulations as a tool to identify factors that influence the survival, growth, and meiotic competence of early stage follicles. The medium formulation that maximized survival and growth was identified as αMEM/F12 supplemented with fetuin, insulin, transferrin, selenium, and follicle stimulating hormone (FSH). This medium stimulated the growth of late primary (average initial diameter of 80 µm) and early secondary (average initial diameter of 90 µm) follicles, which developed antral cavities and increased to terminal diameters exceeding 300 µm in 14 days. Survival ranged from 18% for 80 µm follicles to 36% for 90 µm follicles. Furthermore, 80% of the oocytes from surviving follicles with an initial diameter of 90–100 µm underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 50%. Follicle/oocyte growth and GVBD/MII rates were not significantly different from MEF co-culture. Survival was reduced relative to MEF co-culture, yet substantially increased relative to the control medium that had been previously used for secondary follicles. Continued development of culture medium could enable mechanistic studies of early stage folliculogenesis and emerging strategies for fertility preservation. Biotechnol. Bioeng. 2013;110: 3258–3268. © 2013 Wiley Periodicals, Inc.

Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin β-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LβT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LβT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.

Activin A, a member of the transforming growth factor-β superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-α (TNF-α) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-α, but were unable to respond to LPS directly. Although TNF-α stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-α-induced activin release, and the secretion of activin A was not due to TNF-α-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-α may contribute to the early peak in circulating activin A levels during acute inflammation.

Mature 31- and 34-kDa inhibin A and B negatively regulate the release of FSH from the anterior pituitary; however, a direct comparison of these hormones in vivo has not been undertaken. The bioactivities of highly purified preparations of recombinant human 31-kDa inhibin A and B were determined in rat pituitary cells in vitro, and in ovariectomized adult rats in vivo based on suppression of plasma FSH. The 31-kDa inhibin B was 4.2-fold more bioactive than inhibin A in vitro and 1.45 (1.01-2.79)-fold more bioactive in vivo than 31-kDa inhibin A. However, the corresponding relative binding affinities of 31-kDa inhibin B for betaglycan, betaglycan+activin type II receptor (ActRII)-A, and betaglycan+ActRIIB were lower (IC(50) 2200, 400, and 750 pm, respectively) compared with 31-kDa inhibin A (IC(50) 190, 80, and 290 pm, respectively). A 2.7- and 2.5-fold reduction in in vitro bioactivity was observed between the 31- and 34-kDa inhibin A and 31- and 34-kDa inhibin B, respectively, and these decreases in bioactivities were matched by a parallel reduction in binding to betaglycan and betaglycan+ActRIIA/B. It is concluded that the increased in vitro and in vivo bioactivities of 31-kDa inhibin B cannot be explained by a higher affinity to betaglycan or activin type II receptors; thus, additional factors mediate inhibin B's action. In addition, similar reductions in in vitro bioactivity and betaglycan+ActRIIA/B binding between 31- and 34-kDa inhibins A and B are attributed to hindrance by the additional carbohydrate group at Asn(302) in the formation of a functional inhibin+betaglycan+ActRIIA/B complex.

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases.

Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles, where vascularity is limited. Its role and underlying mechanism in the in vitro activation and maturation of immature to ovulatory follicles is largely unknown. In this study, early secondary (110 μm) murine follicles were isolated and encapsulated in alginate hydrogels to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5 or 20% O2. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% compared with 20% O2. Using a mouse hypoxia-signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of the hypoxia-activated HIF1-complex were significantly upregulated in follicles cultured for 8 days at 2.5% compared with 20% O2, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other upregulated genes included carbohydrate transporters ( Slc2a1, Slc2a3, and Slc16a3) and enzymes essential for glycolysis ( Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2, and Ldha). For follicles cultured at 2.5% O2, a 7.2-fold upregulation of Vegfa correlated to an 18-fold increase in VEGFA levels, and a 3.2-fold upregulation of Ldha correlated to a 4.8-fold increase in lactate levels. Both VEGFA and lactate levels were significantly higher in follicles cultured at 2.5% compared with 20% O2. Therefore, enhanced hypoxia-mediated glycolysis is essential for growth and survival of early secondary follicles and provides vital insights into improving in vitro culture conditions.

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

The in vitro growth of ovarian follicles is an emerging technology for fertility preservation. Various strategies support the culture of secondary and multilayer follicles from various species including mice, non-human primate, and human; however, the culture of early stage (primary and primordial) follicles, which are more abundant in the ovary and survive cryopreservation, has been limited. Hydrogel-encapsulating follicle culture systems that employed feeder cells, such as mouse embryonic fibroblasts (MEFs), stimulated the growth of primary follicles (70–80 μm); yet, survival was low and smaller follicles (

A dynamic partnership between follicle-stimulating hormone (FSH) and activin is required for normal Sertoli cell development and fertility. Disruptions to this partnership trigger Sertoli cells to deviate from their normal developmental pathway, as observed in inhibin α-knockout (Inha-KO) mice, which feature Sertoli cell tumours in adulthood. Here, we identified the developmental windows by which adult Sertoli cell tumourigenesis is most FSH sensitive. FSH was suppressed for 7 days in Inha-KO mice and wild-type littermates during the 1st, 2nd or 4th week after birth and culled in the 5th week to assess the effect on adult Sertoli cell development. Tumour growth was profoundly reduced in adult Inha-KO mice in response to FSH suppression during Weeks 1 and 2, but not Week 4. Proliferative Sertoli cells were markedly reduced in adult Inha-KO mice following FSH suppression during Weeks 1, 2 or 4, resulting in levels similar to those in wild-type mice, with greatest effect observed at the 2 week time point. Apoptotic Sertoli cells increased in adult Inha-KO mice after FSH suppression during Week 4. In conclusion, acute FSH suppression during the 1st or 2nd week after birth in Inha-KO mice profoundly suppresses Sertoli cell tumour progression, probably by inhibiting proliferation in the adult, with early postnatal Sertoli cells being most sensitive to FSH action.

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α-subunit and by an activin-binding protein, follistatin. The activin-β(C) subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin-β(C) forms heterodimers with activin-β(A) and activin-C antagonizes activin-A in vitro. Thus, it is proposed that overexpression, rather than loss of activin-β(C) , regulates activin-A bioactivity. In order to prove biological efficacy, inhibin α-subunit knockout mice (α-KO) were crossed with mice overexpressing activin-β(C) (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin-A, and cancer-associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α-KO/ActC++ mice, where excessive activin-A is the underlying cause. Accordingly, a reduction in activin-A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α-KO/ActC++ mice. Overexpression of activin-β(C) antagonized the activin signalling cascade; thus, the tumourigenic effects of activin-A were abrogated. This study provides proof of the biological relevance of activin-β(C) . Being a regulator of activin-A, it is able to abolish cachexia and modulate reproductive tumour development in α-KO mice.

Elevated activin A levels in inhibin-deficient mice promote the development of gonadal tumors and induce cachexia by reducing muscle, liver, stomach, and fat mass. Because activin A is an important regulator of tissue growth, inhibiting the actions of this TGFβ family ligand may halt or reverse pathology in diseased tissues. In this study, we modified the activin A propeptide to generate a specific activin antagonist. Propeptides mediate the synthesis and secretion of all TGFβ ligands and, for some family members (e.g. TGFβ1), bind the mature growth factor with high enough affinity to confer latency. By linking the C-terminal region of the TGFβ1 propeptide to the N-terminal region of the activin A propeptide, we generated a chimeric molecule [activin/TGFβ1 propeptide (AT propeptide)] with increased affinity for activin A. The AT propeptide was 30-fold more potent than the activin A propeptide at suppressing activin-induced FSH release by LβT2 pituitary gonadotrope cells. Binding of the AT propeptide to activin A shields the type II receptor binding site, thereby reducing Smad2 phosphorylation and downstream signaling. In comparison with the commonly used activin antagonists, follistatin (IC(50) 0.42 nM), soluble activin type II receptor A-Fc (IC(50) 0.47 nM), and soluble activin type II receptor B-Fc (IC(50) 0.91 nM), the AT propeptide (IC(50) 2.6 nM) was slightly less potent. However, it was more specific, inhibiting activin A and activin B (IC(50) 10.26 nM) but not the closely related ligands, myostatin and growth differentiation factor-11. As such, the AT propeptide represents the first specific activin antagonist, and it should be an effective reagent for blocking activin actions in vivo.

Inhibin A and B, dimeric glycoproteins comprising an α- and β((A/B))-subunit, negatively regulate follicle stimulating hormone (FSH) synthesis by the pituitary. The expression of α- and β-subunits within Sertoli cells of the testis and granulosa cells of the ovary is controlled by a range of transcription factors, including CREB, SP-1, Smads, and GATA factors. The inhibin α- and β-subunits are synthesized as precursor molecules consisting of an N-terminal propeptide and a C-terminal mature domain. Recently, we showed that hydrophobic residues within the propeptides of the α- and β-subunits interact noncovalently with their mature domains, maintaining the molecules in a conformation competent for dimerization. Dimeric precursors are cleaved by proprotein convertases and mature inhibins are secreted from the cell noncovalently associated with their propeptides. Propeptides may increase the half-life of inhibin A and B in circulation, but they are readily displaced in the presence of the high-affinity receptors, betaglycan, and ActRII.

Inhibins A and B are gonadal factors that negatively regulate FSH synthesis by the anterior pituitary. Across the menstrual cycle, women show a strong inverse correlation between circulating FSH and inhibin B, estradiol, and anti-Mullerian hormone (AMH), but not with inhibin A. Estradiol is believed to provide a tonic inhibitory effect while the inhibitory role of AMH is unknown. In human males, inhibin B is the primary testicular factor regulating FSH with limited effects by gonadal steroids. In vitro and in vivo studies in rats indicate that inhibin B is more biologically active than inhibin A but showed a lower affinity for the activin type II receptors and the co-receptor, betaglycan, suggesting an alternative mechanism. While this review reinforces the important role inhibin plays in regulating FSH, the observed differences in mode of action of inhibins A and B and their interplay with other gonadal factors are still poorly understood.

Like other members of the transforming growth factor-β (TGF-β) superfamily, activins are synthesised as precursor molecules comprising an N-terminal prodomain and C-terminal mature region. During synthesis, the prodomain interacts non-covalently with mature activin, maintaining the molecule in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases and activin is secreted from the cell non-covalently associated with its propeptide. Extracellularly, the propeptide interacts with heparan sulfate proteoglycans to regulate activin localization within tissues. The mature activin dimer exhibits the classic ‘open-hand’ structure of TGF-β ligands with ‘finger-like’ domains projecting outward from the cysteine knot core of the molecule. These finger domains form the binding epitopes for type I and II serine/threonine kinase receptors. Activins ability to access its signalling receptors is regulated by the extracellular binding proteins, follistatin, follistatin-like-3, and by inhibins, which, in the presence of betaglycan, sequester type II receptors.

Inhibins A and B are gonadal factors that negatively regulate FSH synthesis by the anterior pituitary. Across the menstrual cycle, women show a strong inverse correlation between circulating FSH and inhibin B, estradiol, and anti-Mullerian hormone (AMH), but not with inhibin A. Estradiol is believed to provide a tonic inhibitory effect while the inhibitory role of AMH is unknown. In human males, inhibin B is the primary testicular factor regulating FSH with limited effects by gonadal steroids. In vitro and in vivo studies in rats indicate that inhibin B is more biologically active than inhibin A but showed a lower affinity for the activin type II receptors and the co-receptor, betaglycan, suggesting an alternative mechanism. While this review reinforces the important role inhibin plays in regulating FSH, the observed differences in mode of action of inhibins A and B and their interplay with other gonadal factors are still poorly understood.

Inhibin A and B, dimeric glycoproteins comprising an α- and β((A/B))-subunit, negatively regulate follicle stimulating hormone (FSH) synthesis by the pituitary. The expression of α- and β-subunits within Sertoli cells of the testis and granulosa cells of the ovary is controlled by a range of transcription factors, including CREB, SP-1, Smads, and GATA factors. The inhibin α- and β-subunits are synthesized as precursor molecules consisting of an N-terminal propeptide and a C-terminal mature domain. Recently, we showed that hydrophobic residues within the propeptides of the α- and β-subunits interact noncovalently with their mature domains, maintaining the molecules in a conformation competent for dimerization. Dimeric precursors are cleaved by proprotein convertases and mature inhibins are secreted from the cell noncovalently associated with their propeptides. Propeptides may increase the half-life of inhibin A and B in circulation, but they are readily displaced in the presence of the high-affinity receptors, betaglycan, and ActRII.

Transforming growth factor-beta1 (TGF-beta1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-beta1 propeptide (latency-associated peptide (LAP)), and latent TGF-beta-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-beta1 complex. Initially, hydrophobic LAP residues (Ile(53), Leu(54), Leu(57), and Leu(59)), which form a contiguous epitope on one surface of an amphipathic alpha-helix, interact with mature TGF-beta1 to form the small latent complex. TGF-beta1 binding is predicted to alter LAP conformation, exposing ionic residues (Arg(45), Arg(50), Lys(56), and Arg(58)) on the other side of the alpha-helix, which form the binding site for latent TGF-beta-binding proteins. The stability of the resultant large latent complex is dependent upon covalent dimerization of LAP, which is facilitated by key residues (Phe(198), Asp(199), Val(200), Leu(208), Phe(217), and Leu(219)) at the dimer interface. Significantly, genetic mutations in LAP (e.g. R218H) that cause the rare bone disorder Camurati-Engelmann disease disrupted dimerization and reduced the stability of the latent TGF-beta1 complex.

Background The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development. Methods In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas. Results Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues. Conclusion The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn268 or Asn268 and Asn302 in the α-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 ± 0.15 vs. 0.24 ± 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 ± 0.29) than the 34-kDa form (1.08 ± 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC50, 0.68 nm) than the 34-kDa isoform (IC50, 8.2 nm) at displacing [125I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn302 of the α-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

Activins are members of the TGF-β superfamily that signal via type II and type I receptor subunits and intracellular Smads1. Activin A stimulates FSH release from the pituitary and is also a potent growth and differentiation factor in many physiological systems2. Over-expression of the activin-βC subunit in vitro leads to a reduction in activin A and an increase in activin AC3. Transgenic mice over-expressing activin-βC show decreased circulating activin A, implying that activin AC may also be formed in vivo4. Recently recombinant activin AC has become available, therefore this study examines the in vitro function and mechanism of action of activin AC. Activin AC stimulates FSH release in LβT2 cells and is a negative growth regulator in LNCaP cells, however the potency of activin AC is 8-10 fold less than activin A. Incubation of LNCaP cells with activin receptor antibodies (ALK4, ActRIIA, ActRIIB) abolishes the growth inhibitory effects of activin AC. Activin AC binds to ActRIIB, however a 20-30 fold decrease in both the potency and affinity of activin AC is evident compared to activin A. In addition, activin AC increases Smad-2 phosphorylation. These results indicate activin AC utilises the same receptors and intracellular signalling molecules as activin A. The activin A antagonists, follistatin and activin C4, also antagonise the growth inhibitory effects of activin AC and reduce Smad-2 phosphorylation and Smad-4 expression. This study shows for the first time that the in vitro function of activin AC is similar to activin A, albeit at a lower potency and provides the impetus to determine the physiological role of activin AC in vivo.