Thrombospondin (THBS1) is a large modular glycoprotein component of the extracellular matrix with disulfide-linked subunits and contains a variety of distinct domains, including three repeating subunits (types I, II, and III) that share homology to an assortment of other proteins (Wolf et al., 1990).
This gene is FSH suppressed. Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization. Bonnet A et al. ABSTRACT: FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts. A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin.
NCBI Summary:
The protein encoded by this gene is a subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. This protein can bind to fibrinogen, fibronectin, laminin, type V collagen and integrins alpha-V/beta-1. This protein has been shown to play roles in platelet aggregation, angiogenesis, and tumorigenesis. [provided by RefSeq, Jul 2008]
General function
Cell adhesion molecule
Comment
Frazier (1987) reviewed the molecular structure of thrombospondin. THBS was first described as a component of the alpha-granule of platelets, released on platelet activation. It is associated with the platelet membrane in the presence of divalent cations and has a role in platelet aggregation. THBS is not limited to platelets, however. It is synthesized and secreted for incorporation into the extracellular matrix by a variety of cells including endothelial cells, fibroblasts, smooth muscle cells, and type II pneumocytes. THBS binds heparin, sulfatides, fibrinogen, fibronectin, plasminogen, and type V collagen.
Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1).
J Cell Physiol. 2006 .
Cellular localization
Secreted
Comment
Chronic effects of an anti-angiogenic thrombospondin-1 mimetic peptide, ABT-898, on female mouse reproductive outcomes. Edwards AK et al. (2016) Angiogenesis is an essential process in endometriosis disease progression. Earlier, we demonstrated that anti-angiogenic peptide, ABT-898 prevents neoangiogenesis of human endometriotic lesions in a xenograft mouse model. Since angiogenesis is essential for normal ovarian and uterine function, we evaluated effects of ABT-898 on normal female reproductive processes in mice. Cycling female C57BL/6N mice were dosed with ABT-898 (100 mg/kg) or 5 % dextrose control for 21 consecutive days to cover multiple estrous cycles (average estrous cycle 4 to 5 days in mice). Pregnant female mice were dosed with ABT-898 (100 mg/kg) or control on alternate days over the course of gestation, beginning at gestation day 7.5 to 17.5 (gestation length 21 days). Histological analysis along with CD31 and Vimentin immunohistochemistry were performed on ovaries and uteri obtained from treated and control mice. To understand the influence of ABT-898 on systemic angiogenic factors, a Pro Mouse Cytokine 9-plex assay was performed on plasma samples obtained from mice prior to treatment, during the second week of ABAT-898 or control treatment and on the last day of treatment. ABT-898 did not affect the number of estrous cycles over the 21 day treatment compared to control. Histological analysis of ovaries found no difference in the number of primordial, primary, secondary, and antral follicles between ABT-898 treated and control groups. Similarly, no difference was observed in the microvessel density between ABT-898 treated and control uteri, ovarian follicles or corpus luteum when assessed using CD31 or vimentin immunohistochemistry. Electron microscopy revealed similar capillary structure and appearance in both ABT-898 treated and control uteri. Although peripheral blood angiogenic cytokine profiles (IL-15, IL-18, M-CSF, b-FGF, PDGF-bb, MIG, MIP-2, LIF and VEGF) changed over the course of the intervention, there was no significant difference between ABT-898 and control groups at any of the studied time points. Treatment with ABT-898 during pregnancy had no effect on litter size at birth, pup weight at birth or pup weight at weaning. Our findings suggest that ABT-898 may not alter angiogenesis dependent reproductive processes in female mice. However, an extensive reproductive toxicology screening is required to substantiate use of ABT-898 in future.//////////////////
Thrombospondin 1 (THBS1) Promotes Follicular Angiogenesis, Luteinization, and Ovulation in Primates. Bender HR et al. (2019) Angiogenesis is essential to both ovulation and the formation of the corpus luteum. The thrombospondin (THBS) family of glycoproteins plays diverse roles in regulation of angiogenesis, but the role of these vascular regulators in ovulation and luteinization remain to be elucidated. Using the cynomolgus macaque as a model for human ovulation, we demonstrated that levels of THBS1 mRNA and protein in preovulatory follicle granulosa cells increased after the ovulatory gonadotropin surge, with peak levels just before the expected time of ovulation. THBS1 treatment of monkey ovarian microvascular endothelial cells in vitro stimulated migration, proliferation, and capillary sprout formation, consistent with a pro-angiogenic action of THBS1. Injection of an anti-THBS1 antibody into monkey preovulatory follicles reduced rates of follicle rupture and oocyte release in response to an ovulatory gonadotropin stimulus when compared with control IgG-injected follicles. Interestingly, two of three oocytes from anti-THBS1 antibody injected follicles were germinal vesicle intact, indicating that meiosis failed to resume as anticipated. Follicles injected with anti-THBS1 antibody also showed reduced granulosa cell layer expansion, endothelial cell invasion, and capillary formation when compared to control IgG-injected follicles. Overall, these findings support a critical role for THBS1 in follicular angiogenesis, with implications for both successful ovulation and corpus luteum formation.//////////////////
Zinc finger gene 217 (ZNF217) Promoted Ovarian Hyperstimulation Syndrome (OHSS) through Regulating E2 Synthesis and Inhibiting Thrombospondin-1 (TSP-1). Zhai J et al. (2017) Zinc finger gene 217 (ZNF217) is a candidate gene of polycystic ovary syndrome (PCOS) which is vulnerable to ovarian hyperstimulation syndrome (OHSS). However, the relationship between ZNF217 and OHSS is largely unknown. Our study demonstrated that ZNF217 was mainly distributed in the granulosa cells of rat ovary. Significantly higher expression of ovarian ZNF217 was detected in OHSS rats, being consistent with serum 17β-estradiol concentration and ovarian aromatase. Moreover, OHSS rats also showed decreased ovarian TSP-1 mRNA, an acknowledged VEGF signaling suppressor. The same changes were detected in human granulosa cells and follicular fluid. Thus, the increased ZNF217 and decreased TSP-1 may participate in OHSS onset. In vitro experiment revealed that ZNF217 positively regulated E2 synthesis through promoting cAMP response element binding protein (CREB) and thereby CYP19A1 in KGN cells. Furthermore, ZNF217 negatively regulated TSP-1 in KGN cells while TSP-1 promoted claudin1 and inhibited nitric oxide (NO) in HUVECs and HAECs. Both of claudin1 and NO are responsible for the regulation of vascular permeability (VP). Therefore, we demonstrated that ZNF217 contributed to OHSS onset through promoting E2 synthesis and the increase of VP. Moreover, the increased ZNF217 and decreased TSP-1 provided new targets for the prevention or treatment of OHSS in the future.//////////////////
Thrombospondin-1 Affects Bovine Luteal Function via Transforming Growth Factor-Beta1-Dependent and Independent Actions. Farberov S et al. (2015) Thrombospondin-1 (THBS1) and transforming growth factor-beta1 (TGFB1) are specifically up-regulated by prostaglandin F2alpha in mature CL. This study examined the relationship between the expression of THBS1 and TGFB1 and the underlying mechanisms of their actions in luteal endothelial cells (EC). TGFB1 stimulated SMAD2 phosphorylation and SERPINE1 levels in dose- and time-dependent manners in luteal EC. THBS1 also elevated SERPINE1, this effect was abolished by TGFB1 receptor-1 kinase inhibitor (SB431542). The findings below further imply that THBS1 activates TGFB1 in luteal EC: THBS1 increased the effects of latent TGFB1 on phospho-SMAD2 and SERPINE1. THBS1 silencing significantly decreased SERPINE1 and levels of phospho-SMAD2. Lastly, THBS1 actions on SERPINE1 were inhibited by LSKL peptide (TGFB1 activation inhibitor); LSKL also counteracted latent TGFB1-induced phospho-SMAD2. We found that TGFB1 up-regulated its own mRNA levels and those of THBS1. Both compounds generated apoptosis, but THBS1 was significantly more effective (2.5-fold). Notably, this effect of THBS1 was not mediated by TGFB1. THBS1 and TGFB1 also differed in their activation of p38 MAPK. Whereas TGFB1 rapidly induced phospho-p38, THBS1 had a delayed effect. Inhibition of p38 pathway by SB203580 did not modulate TGFB1 effect on cell viability, but it amplified THBS1 actions. THBS1-stimulated caspase-3 activation coincided with p38 phosphorylation, suggesting that caspase-induced DNA damage initiated p38 phosphorylation. The in vitro data suggest that a feed-forward loop exists between THBS1, TGFB1, and SERPINE1. Indeed all these three genes were similarly induced in the regressing CL. Their gene products can promote vascular instability, apoptosis, and matrix remodeling during luteolysis.//////////////////
Thrombospondin-1 Inhibits Angiogenesis and Promotes Follicular Atresia in a Novel in Vitro Angiogenesis Assay. Garside SA et al. Thrombospondin-1 (TSP-1) is a putative antiangiogenic factor, but its role in regulating physiological angiogenesis is unclear. We have developed a novel in vitro angiogenesis assay to study the effect of TSP-1 on follicular angiogenesis and development. Intact preantral/early antral follicles dissected from 21-d-old rat ovaries were cultured for 6 d in the presence or absence of TSP-1. At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. Follicles were fixed and sectioned, and follicular apoptosis was assessed by immunohistochemistry for activated caspase-3 in granulosa cells. The results showed that TSP-1 inhibited follicular angiogenesis (P < 0.01) and promoted follicular apoptosis (P < 0.001) in a dose-dependent manner. To determine whether the proapoptotic activity of TSP-1 is mediated by direct effects on granulosa cells, isolated granulosa cells were cultured with TSP-1 (0, 10, 100, and 1000 ng/ml) for 48 h. Apoptosis was quantified using a luminescent caspase-3/7 assay. TSP-1 promoted apoptosis of granulosa cells in a dose-dependent manner (P < 0.05), suggesting that TSP-1 can act independently of the angiogenesis pathway to promote follicular apoptosis. These results show that TSP-1 can both inhibit follicular angiogenesis and directly induce apoptosis of granulosa cells. As such, it may have potential as a therapeutic for abnormal ovarian angiogenesis and could facilitate the destruction of abnormal follicles observed in polycystic ovary syndrome.
Bagavandoss et al. (1998) characterized the expression of two secreted matricellular proteins associated with angiogenesis, SPARC and thrombospondin (TSP), in postovulatory preluteal follicles and corpus luteum (CL) of hormone-primed immature rats. By indirect immunofluorescence with specific antibodies, TSP was prominent in the basement membranes of growing follicles. Many cells in the early vascularizing corpus luteum (CL) also expressed TSP. Neovascularization of CL was accompanied by expression of TSP in central avascular areas. In mature CL, steroidogenic luteal cells expressed TSP but regressing CL did not express TSP. The observed changes in expression of TSP during development of the CL support distinct roles in nonpathological morphogenesis and angiogenesis.
Thrombospondin-1 Expression is increased during Follicular Atresia in the Primate Ovary. Thomas FH et al. Thrombospondin (TSP)-1 is an anti-angiogenic extracellular matrix glycoprotein that modulates several aspects of cellular function. The aim of this study was to determine the pattern of TSP-1 mRNA and protein expression, as well as expression of its receptor CD36 in the marmoset ovary, and to investigate the effects of inhibition of gonadotropins or VEGF activity on TSP-1 and CD36 expression in vivo. GnRH antagonist or VEGF Trap, a soluble decoy receptor, was administered on Day 0 of the follicular phase of the cycle, and ovaries were collected at the end of the follicular phase (Day 10). TSP-1 mRNA and protein was present in granulosa cells of preantral and antral follicles, with the highest staining at the late secondary and tertiary stages. Moreover, expression of TSP-1 mRNA and protein was significantly increased in tertiary follicles undergoing atresia. CD36 protein was detected in granulosa cells of preantral and antral follicles, as well as in endothelial cells of large vessels. Inhibition of gonadotropin secretion or VEGF activity had no effect on TSP-1 expression; however, expression of CD36 protein was inhibited by the VEGF Trap. In conclusion, TSP-1 may be involved in the cessation of angiogenesis in follicles undergoing atresia; alternatively, TSP-1 may act on granulosa and/or endothelial cells to promote follicular atresia in the ovary. Angiogenesis is likely to involve a balance between pro- and anti-angiogenic factors. Our results suggest that loss of VEGF activity does not regulate TSP-1 expression directly, but may influence TSP-1 activity via downregulation of the CD36 receptor.
A Thrombospondin-Mimetic Peptide, ABT-898, Suppresses Angiogenesis and Promotes Follicular Atresia in Pre- and Early-Antral Follicles in Vivo. Garside SA et al. Using a novel in vitro angiogenesis assay, we previously showed that thrombospondin (TSP)-1 has antiangiogenic effects on rat follicles and induces apoptosis in granulosa cells in vitro. ABT-898 is an octapeptide mimetic of TSP-1 closely related to ABT-510. Here, we demonstrate the inhibitory effects of ABT-898 on follicular angiogenesis and its proapoptotic effect on granulosa cells. To investigate the potential of this peptide to inhibit follicular angiogenesis in vivo, marmoset monkeys were treated with 2.5 mg/kg ABT-898 twice daily throughout the follicular phase of the cycle. Although treatment did not block emergence of dominant follicles, angiogenesis was reduced in preantral and early-antral follicles. Furthermore, the incidence of atresia at these follicle stages was increased. To investigate whether treatment with ABT-898 would interfere with the timing or duration of the normal ovulatory rise in plasma progesterone, marmosets were treated with a depot formulation containing 25 mg ABT-898 at the start of the follicular phase, with a second injection after 2 wk. Despite active concentrations of peptide being maintained in the circulation, no apparent effects on the ovulatory cycle were observed. Taken together, these results indicate that ABT-898 is capable of having a dual effect by inhibiting follicular angiogenesis and promoting atresia of antral follicles in vivo but does not prevent ovulation or induce luteolysis, as has been observed with direct vascular endothelial growth factor inhibitors. These results suggest that ABT-898 could be a novel therapeutic to inhibit abnormal angiogenesis and induce atresia of accumulated follicles in polycystic ovary syndrome.
FSH promotes the proliferation of sheep granulosa cells by inhibiting the expression of TSP1. Li X et al. (2020) Thrombospondin (TSP1) plays an important role as an antiangiogenic factor in the reproductive system of female mammals. However, its expression and function in sheep are still unclear. In the present research, the Altay sheep (a native Chinese breed) was used to analyze the expression of TSP1 in the ovary and its potential function in granulosa cells. TSP1 was widely expressed in most tissues, as shown by qPCR. In the ovary, TSP1 mRNA expression decreased during follicular to luteal growth. The TSP1 protein was expressed in a wide variety of follicles of different diameters and localized to the cytoplasm and nucleus of granulosa cells. In in vitro studies, follicle-stimulating hormone (FSH) significantly inhibited the expression of TSP1 in sheep granulosa cells. Functionally, FSH- and TSP1-specific siRNAs can promote the proliferation of sheep granulosa cells. In contrast, TSP1 mimetic peptide, ABT510, offsets the proliferation of sheep granulosa cells. Different signaling pathway inhibitors all promoted FSH-inhibited TSP1 expression, but each inhibitor had different effects on TSP1. Among them, the PI3K and ERK pathway inhibitors significantly promoted TSP1 expression and inhibited the proliferation of sheep granulosa cells.////////////////// MiR-222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene. Zhu W et al. (2019) Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA-222 (miR-222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR-222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR-222 functions as an anti-apoptotic factor in pGCs. MiR-222 mimics in pGCs result in the upregulation of the anti-apoptotic BCL-2 gene, down-regulation of the proapoptotic caspase-3 gene, and inhibition of apoptosis. MiR-222 inhibitors reduced BCL-2 and had no significant effect on caspase-3. MiR-222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1-siRNA reduced the proapoptotic BAX gene. MiR-222 can directly target the 3'-untranslated region of the THBS1 gene. MiR-222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR-222 inhibitor. Transfection of THBS1-siRNA resulted in the inhibition of the miR-222 inhibitor, which suggests that miR-222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR-222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.////////////////// Fibroblast Growth Factor-2 and Transforming Growth Factor-beta1 Oppositely Regulate miR-221 that Targets Thrombospondin-1 in Bovine Luteal Endothelial Cells. Farberov S et al. (2017) Thrombospondin-1 (THBS1) is an important mediator of corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. In this study we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced SERPINE1 in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.//////////////////
Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary. Berisha B et al. (2016) The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and > 180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16, and > 18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7, > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.//////////////////
Regulation of Angiogenesis-Related Prostaglandin
F2alpha-Induced Genes in the Bovine Corpus Luteum. Zalman Y et al. We recently compared prostaglandin F2alpha (PG)-induced global gene expression profiles in PG-refractory, bovine corpus luteum (CL) collected on day (d) 4 of the estrous cycle, versus PG-responsive, d11 CL. Transcriptome analyses led us to study the regulation of angiogenesis-related genes by PG and their functions in luteal endothelial cells (ECs). We found that PG regulated angiogenesis-modulating factors in a luteal stage-dependent way. A robust increase in FGF2 expression (mRNA and protein) occurred in the PG-refractory d4 CL promoting CL survival and function. Inhibitors of FGF2 action, thrombospondin 1 and 2, their receptor (CD36), and PTX3 were upregulated by PG specifically in d11 CL undergoing luteolysis. VEGF mRNA decreased 4h post PG in both d4 and d11 CL. The resulting destabilization of blood vessels in d11 CL is expected to weaken the gland and reduce its hormonal output. These genes were expressed in dispersed luteal ECs and steroidogenic cells, however, thrombospondin 1 and FGF2 were more abundant in luteal ECs. Expression of such genes and their ability to modulate FGF2 actions were investigated. Similarly to its in vivo effect, PG stimulated in vitro the expression of thrombospondins and PTX3 genes in several luteal cell models. Importantly, these factors influenced the angiogenic properties of luteal ECs. FGF2 dose-dependently enhanced cell migration and proliferation, whereas thrombospondin 1 and PTX3 inhibited FGF2 actions in luteal ECs. Collectively, the data presented here suggest that by tilting the balance between pro- and anti-angiogenic factors, PG can potentially control the ability of the CL to resist or advance toward luteolysis.
Rapid Insulin-like Growth Factor-1-induced Changes in Granulosa Cell Thrombospondin-1 Expression In Vitro. McGray AJ et al. Thrombospondin-1 (TSP-1) is a large extracellular matrix-associated protein that is important for normal follicular development, is rapidly modulated during follicular growth and plays important roles in cellular proliferation and angiogenesis. TSP-1 mRNA is post-transcriptionally regulated, although the underlying mechanisms are largely unknown. Insulin-like growth factor-1 is a potent signalling molecule that participates in folliculogenesis. We hypothesized that IGF-1 modulates TSP-1 expression in granulosa cells, and that such modulation requires rapid turnover of the TSP-1 mRNA and protein. Spontaneously-immortalized rat granulosa cells (SIGC) were cultured in the presence or absence of IGF-1, after which the expression and turnover of TSP-1 mRNA and protein was evaluated by western blot and quantitative PCR. RNA stability reporter constructs were prepared in which wild-type and mutated AU-rich elements from the TSP-1 3'UTR were cloned downstream of the luciferase gene in a mammalian expression vector. These were transfected into SIGC cells in order to characterize mRNA elements that regulate the stability of the TSP-1 mRNA. TSP-1 expression decreased rapidly at the mRNA and protein levels in IGF-1 treated cultures. Following 12 h of IGF-I treatment, TSP-1 protein decreased by 25% and was 73% lower than in untreated cultures. The half-life of endogenous TSP-1 mRNA in SIGC was 2.0 h. This was not changed in the presence of IGF-1, however, transcription of new TSP-1 mRNA was inhibited. Reporter mRNAs with mutated AU-rich elements demonstrated a longer half-life than mRNAs in which the wild type AU-rich elements were present. These studies reveal that IGF-1 rapidly inhibits TSP-1 expression at the protein and mRNA levels in cultured granulosa cells through apparent inhibition of TSP-1 transcription. The decrease depends on an intrinsically short half-life of TSP-1 mRNA and protein. The short mRNA half life is due, at least in part, to AU-rich elements in the 3'UTR of the TSP-1 mRNA.
Dreyfus et al. (1992) studied the hormonal effect of TSP production and receptor-mediated binding to primary granulosa cells prepared from diethylstilbestrol-treated immature female rats. Undifferentiated granulosa cells synthesize and secrete TSP. The protein comprises about 0.5% of the total cell protein, and it is the major protein secreted in culture. Treatment of the cells with FSH or 8-Br-cAMP reduces TSP production dramatically, and forskolin completely inhibits it. In parallel, the undifferentiated granulosa cells bind TSP specifically with a Kd of 1.8 nM, and the number of binding sites per cell is 1.7 x 10(5). This binding can be prevented by excess TSP or an anti-TSP monoclonal antibody (B7-3). This ability to bind TSP is completely lost after induction of differentiation by FSH or 8-Br-cAMP. Dreyfus et al. (1992)concluded that both the production and binding of TSP to granulosa cells are tightly controlled by normal cell differentiation and indicate that changes in TSP are correlated with the passage of the cell through the stages of maturation, a passage that also involves changes in cell shape and extracellular interactions and in the steroidogenic capacity of these cells.
Microarray analysis of insulin-like growth factor-I-induced changes in mRNA expression in cultured porcine granulosa cells: Possible role of IGF-I in angiogenesis. Grado-Ahuir JA et al. IGF-I in conjunction with gonadotropins are important stimulators of mitosis and ovarian steroid production by granulosa and thecal cells, which are required for normal oocyte development and hormonal feedback signaling to the hypothalamus and pituitary. However, a comprehensive evaluation of the changes in gene expression induced by IGF-I has not been conducted. Our objective was to characterize granulosa cell gene expression in response to IGF-I treatment. Porcine granulosa cells were pooled in 4 biological replicates and treated with FSH (baseline) or FSH+IGF-I for 24 h in vitro. The RNA was collected and hybridized to 8 Affymetrix Porcine GeneChips (Affymetrix, Santa Clara, CA) in a paired design. Differentially regulated gene sequence element sets (P < 0.01) were used as queries in the UniGene database searching for annotated genes. Abundance of mRNA for genes differentially expressed in the microarray analysis was determined through multiplex assays of one-step real-time RT-PCR and further analyzed under a statistical model including the fixed effect of treatment. A total of 388 gene sequence element sets were differentially expressed and 42 matched annotated genes in the UniGene database. Of the 3 upregulated target genes selected for further quantitative RT-PCR analysis, only FGF receptor 2 III c (FGFR2IIIc) mRNA abundance was significantly increased by IGF-I. Of the 3 down-regulated target genes selected for further analysis, only thrombospondin-1 (THBS1) mRNA abundance was significantly decreased by IGF-I. Further study revealed that neither FSH nor estradiol affected the IGF-I-induced suppression of THBS1 mRNA abundance. These results provide the first comprehensive assessment of IGF-I-induced gene expression in granulosa cells and will contribute to a better understanding of the molecular mechanisms of IGF-I regulation of follicular development. Involvement of FGFR2IIIc and THBS1 in mediating IGF-I-induced granulosa cell steroidogenesis and proliferation during follicular development is novel, but their specific roles will require further elucidation.
Ovarian localization
Granulosa, Luteal cells
Comment
Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary. Berisha B et al. (2016) The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.//////////////////
Higuchi et al. (1999) reported the expession of thrombospondin-1 (TSP-1), which is a ligand for CD47, in human granulosa cell (GC) by immunocytochemistry and RT-PCR. Co-expresson of CD47 and TSP-1 on human GC may suggest that TSP-1 has a physiological role in GC function possibly via CD47 in an autocrine fashion.
Follicle stages
Secondary, Antral, Preovulatory, Corpus luteum
Comment
Thrombospondin and Vascular Endothelial Growth Factor are Cyclically Expressed in an Inverse Pattern During Bovine Ovarian Follicle Development Greenaway J, et al .
Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and anti-angiogenic factors. The purpose of this study was to determine the expression of the anti-angiogenic thrombospondin family and pro-angiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size (<0.5cm - small, 0.5-1.0cm - medium, >1.0cm - large). Thrombospondin protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation, and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP co-localized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast to TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and Localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.
Phenotypes
POF (premature ovarian failure)
Mutations
2 mutations
Species: human
Mutation name: type: naturally occurring fertility: subfertile Comment: New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing. Patiño LC et al. (2017) Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. This is a retrospective cohort study performed on 69 women affected by POI. WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest.//////////////////
Species: mouse
Mutation name: type: null mutation fertility: fertile Comment: Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. Lawler J et al. (1998) The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.//////////////////