Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo

TGFbeta/activin/Nodal receptors activate both Smad2 and Smad3 intracellular effector proteins. The functional activities of these closely related molecules have been extensively studied in cell lines. We show both are expressed in the early mouse embryo from the blastocyst stage onwards and mediate Foxh1-dependent activation of the Nodal autoregulatory enhancer in vitro. Genetic manipulation of their expression ratios reveals that Smad3 contributes essential signals at early post-implantation stages. Thus, loss of Smad3 in the context of one wild-type copy of Smad2 results in impaired production of anterior axial mesendoderm, while selective removal of both Smad2 and Smad3 from the epiblast additionally disrupts specification of axial and paraxial mesodermal derivatives. Finally, we demonstrate that Smad2;Smad3 double homozygous mutants entirely lack mesoderm and fail to gastrulate. Collectively, these results demonstrate that dose-dependent Smad2 and Smad3 signals cooperatively mediate cell fate decisions in the early mouse embryo.     

Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.     

Smad2 and Smad3 coordinately regulate craniofacial and endodermal development

Ligands of the transforming growth factor-beta (TGF-beta) superfamily are involved in numerous developmental and disease processes. TGF-beta, activins, and nodal ligands operate through the highly homologous Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at a full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior and ventral midline are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression as well as a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial and midline development, as well as hepatogenesis.     

Smad signalling in the ovary

It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.     

The effects of progesterone on follicular growth in the rabbit ovary

Studies were undertaken to measure the growth of follicles in the rabbit ovary during periods of elevated blood levels of progesterone. The progestin was increased in the blood by pregnancy or by implantation of progesterone pellets, which raised blood progesterone to near the levels measured during pregnancy. After 1, 2, 3, or 4 weeks of pregnancy or progesterone-pellet treatment, follicles of 1.0 mm external diameter or greater were dissected out of the ovaries and their external diameters were measured; then, each follicle was extracted for measurement of estradiol content. Blood levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in these animals as well. Follicles up to 2.5 mm in diameter were found in the ovaries of nonpregnant and untreated animals while 1.8 mm was the maximal size found during pregnancy or progesterone-pellet treatment. Furthermore, both in pregnant and in progesterone-treated rabbits, the follicular estradiol content and concentration were significantly suppressed compared to follicles from untreated rabbits. The progesterone pellets had no major effect on the levels of LH and FSH in the blood; the concentration of these gonadotropins in the progesterone-treated rabbits was virtually identical to levels previously measured in the blood of pregnant animals. The results of these studies indicate that progesterone exerts an inhibitory action on follicular development and steroidogenic function in the rabbit ovary. The progesterone action appears to be exerted directly on the ovary and is not indirect, by way of an inhibition of gonadotropin secretion.     

The number of clonal precursors of the follicular epithelium in the mouse ovary

The number of clonal precursors of granulosa cells in mouse ovarian follicles has been estimated using a technique based on the phenomenon of random X-chromosome inactivation of somatic cells and the use of an X-linked alloenzyme variant of the glycolytic enzyme PGK-1. The granulosa cells of follicles were oligoclonal in origin and founded by a small number of cells (about 5) which was consistent with histological observations. When the analysis was extended to two subcompartments of the follicle, the mural and cumulus granulosa cells, the results indicated that the cumulus and mural granulosa cells had a common origin.     

Syndecan-4 expression is associated with follicular atresia in mouse ovary

Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.     

Retention of fibroblast growth factor 3 in the Golgi complex may regulate its export from cells.

The fibroblast growth factors (FGFs) fall into two distinct groups with respect to their mode of release from cells. Whereas FGF1 and FGF2 lack conventional signal peptides, the remaining members have typical features of secreted proteins. However, the behavior of mouse FGF3 is anomalous, since, despite entering the secretory pathway and undergoing primary glycosylation, its release from transfected COS-1 cells is very inefficient compared with that of FGF4 and FGF5. To investigate the unusual properties of FGF3, we analyzed the processing, secretion, and intracellular localization of a series of site-directed mutants as well as chimeras produced by fusing parts of FGF3, FGF4, and FGF5. Wild-type FGF3 was shown to accumulate in an immature form in the Golgi complex, from where it is slowly released into the extracellular matrix. Removing or relocating the Asn-linked glycosylation site further impaired its release, and exchanging the signal peptide or carboxy terminus had little effect. In contrast, a chimeric protein with an amino terminus from FGF5 was efficiently secreted and biologically active in cell transformation assays. The data suggest that a structural feature of FGF3 involving the amino-terminal region and glycosylation site has a significant bearing on its passage through the Golgi complex and may regulate the secretion of the ligand.     

Theca cells may be present at the outset of follicular growth

Opinions differ concerning when theca cells arise during follicular growth. Theca cells cannot be distinguished from ovarian stroma cells on the basis of morphology alone. However, long-term continuous infusion of [3]thymidine (3H-TdR) thoroughly labels theca cells of large and medium-sized follicles whereas most stromal cells remain unlabeled. The objective of this study was to determine when, during the course of follicular development, labeled, squamous cells located just outside the follicular basement membrane (putative theca cells) first become apparent. Rats were given infusions of 3H-TdR for various periods of time. Autoradiographs were prepared from 2-microns-thick plastic sections of ovaries. Morphologically recognizable theca cells of large and medium-sized follicles were sharply delineated from surrounding stroma cells by their labeling pattern. Although unilaminar follicles lacked morphologically distinct theca layers, they all were closely enveloped by labeled cells just outside the basement membrane. Follicles with a few as 4-5 granulosa cells in cross section often had labeled cells on the convex surface of their basement membranes. These observations suggest the possibility that theca cells may already be present at the very outset of follicular growth.     

GM3 ganglioside induces hamster fibroblast growth inhibition in chemically-defined medium: ganglioside may regulate growth factor receptor function

GM₃ or GM₁ ganglioside exogenously added in chemically-defined medium incorporate equally into cells. However, only GM₃ showed a significant growth inhibition to hamster fibroblasts (BHK). The GM₃-fed cells became refractory to growth stimulation by fibroblast growth factor (FGF) in chemically-defined media. Radiolabeled FGF accumulated on GM₃-fed cells, but not on GM₁-fed cells. Both GM₃ and GM₁ did not directly interact with FGF. These data suggest that GM₃ may regulate the function of the receptors for growth hormones.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.     

Distribution and Y397 phosphorylation of focal adhesion kinase on follicular development in the mouse ovary

Several protein tyrosine kinases (PTKs) are identified as follicle survival factors that suppress apoptosis in granulosa cells. Focal adhesion kinase (FAK/PTK2) interacts with numerous signaling partners and is important for cell adhesion, survival and other vital processes in which FAK autophosphorylation at Y397 (pY397 FAK) is critical for activating signaling pathways. Despite its important roles in apoptosis, the expression and function of FAK in the ovaries remain unknown. Here, we describe FAK expression, including pY397 FAK, in normal healthy mouse ovaries and its association with follicular development and/or atresia. Normal healthy mouse ovaries were used for western blot (n > 60) and immunohistochemical (n > 180) analyses. Western blot results in immature and mature mice revealed that total FAK and pY397 FAK were highly expressed in the ovary and immunohistochemistry results in 3-week-old mice showed they were localized to granulosa cells of ovarian follicles, especially preantral follicles. In 3-week-old mice treated with 5 IU pregnant mare serum gonadotropin (for obtaining homogenous populations of growing or atretic follicles), western blotting revealed that follicular atresia progression involved decreased phosphorylation of Y397 at 72 and 96 h after treatment, particularly in granulosa cells of atretic follicles, as shown by immunohistochemistry results at 72 h after treatment. Moreover, immunostaining patterns of FAK and cleaved caspase-3 were negatively correlated in serial sections of 3-week-old mouse ovaries. These results suggest that FAK is most active in ovarian follicle granulosa cells and that its phosphorylation at Y397 is histologically meaningful in follicular development in normal healthy ovaries.