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Notch signalling regulates steroidogenesis in mouse ovarian granulosa cells. Wang Y et al. (2019) The Notch signalling pathway in the mammalian ovary regulates granulosa cell proliferation. However, the effects of Notch signalling on steroidogenesis are unclear. In this study we cultured mouse ovarian granulosa cells from preantral follicles invitro and observed the effect of Notch signalling on steroidogenesis through overexpression, knockdown and inhibition of Notch signalling. Activation of Notch signalling decreased progesterone and oestrogen secretion. In contrast, inhibition of Notch signalling increased the production of progesterone and oestrogen. Expression of the genes for steroidogenic-related enzymes, including 3β-hydroxysteroid dehydrogenase, p450 cholesterol side-chain cleavage enzyme and aromatase, was repressed after stimulation of Notch signalling. The expression of upstream transcription factors, including steroidogenic factor 1 (SF1), Wilms' tumour 1 (Wt1), GATA-binding protein 4 (Gata4) and Gata6, was also inhibited after stimulation of Notch signalling. Production of interleukin (IL)-6 was positively correlated with Notch signalling and negatively correlated with the expression of these transcription factors and enzymes. In conclusion, Notch signalling regulated progesterone and oestrogen secretion by affecting the expression of upstream transcription factors SF1, Wt1, Gata4 and Gata6, as well as downstream steroidogenic-related enzymes. IL-6, which may be regulated directly by Notch signalling, may contribute to this process. Our findings add to the understanding of the diverse functions of Notch signalling in the mammalian ovary.//////////////////
ADAM10-Notch signaling governs the recruitment of ovarian pregranulosa cells and controls folliculogenesis in mice. Feng L et al. (2016) Ovarian follicles are the basic functional units of female reproduction in the mammalian ovary. We show here that A Disintegrin and Metalloproteinase Domain 10 (ADAM10), a cell surface sheddase, plays an indispensable role in controlling primordial follicle formation by regulating the recruitment of follicle supporting cells in mice. We demonstrate that suppressing ADAM10in vitroor deletionAdam10 in vivodisrupts germline cyst breakdown and primordial follicle formation. Using a cell lineage tracing approach, we show that ADAM10 governs the recruitment of ovarian follicle cells by regulating the differentiation and proliferation of LGR5(+)follicle supporting progenitor cells. By detecting the development of FOXL2(+)pregranulosa cells, we found that inhibiting ADAM10 reduced the number of FOXL2(+)cells in perinatal ovaries. Furthermore, inhibiting ADAM10 suppressed the activation of Notch signaling, and blocking Notch signaling also disrupted the recruitment of follicle progenitor cells. We found that ADAM10-Notch signaling in ovarian somatic cells governs the primordial follicle formation by controlling the development of ovarian pregranulosa cells. The proper recruitment of ovarian follicle supporting cells is essential for the establishment of ovarian reserve in mice.//////////////////
Role of Notch signaling in granulosa cell proliferation and polyovular follicle induction during folliculogenesis in mouse ovary. Terauchi KJ et al. (2016) In the fetal mouse ovary, oocytes are connected by an intercellular bridge and form germ cell cysts. Folliculogenesis begins after birth. To study the role of Notch signaling in folliculogenesis, double-immunohistochemical localization of laminin and Ki-67 was performed in mouse ovaries from embryonic day 17.5 (E17.5) to postnatal day 4 (P4). Most cysts and follicles contained Ki-67-negative cells; however, a few Ki-67-positive cells were present in cysts from E17.5 through P4, indicating that a small number of pre-granulosa cells continue to proliferate during folliculogenesis. To examine the effects of an inhibitor of Notch signaling (DAPT) and a synthetic estrogen (diethylstilbestrol DES]) on folliculogenesis, an organ-culture system was established. The numbers of cysts, primordial follicles (PrFs) and primary follicles were unchanged by DES, whereas the total number of PrFs and of PrFs with Ki-67-negative cells was reduced by DAPT. In organ-cultured neonatal ovaries, only DAPT treatment increased degenerating cells defined as oocytes. On the contrary, the number of polyovular follicles (PFs) and the PF incidence were significantly increased in ovaries organ-cultured with DES at day 20 post-grafting. In organ-cultured fetal and neonatal ovaries, DAPT reduced Notch 3 and Hey2 mRNAs, whereas DES increased Hey2 mRNA. These results suggest that Notch signaling in fetal ovaries is involved with PrF assembly by the regulation of oocyte survival rather than by cell proliferation. In PF induction, as a result of the disruption of interactions between oocytes and pre-granulosa cells, DES and Notch signaling act independently.//////////////////
Notch Signaling Pathway Regulates Progesterone Secretion in Murine Luteal Cells. [Wang J et al. (2015) Notch signaling is an evolutionarily conserved pathway, which involves in various cell life activities. Other studies and our report showed that the Notch signaling plays very important role in follicle development in mammalian ovaries. In luteal cells, Notch ligand, delta-like ligand 4, is involved in normal luteal vasculature. In this study, murine luteal cells were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT). We found that L-658,458 and DAPT treatment decrease basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion. On the contrary, overexpression of intracellular domain of Notch3 increased basal and hCG-stimulated progesterone secretion. Further studies demonstrated that Notch signaling regulated the expression of steroidogenic acute regulatory protein and CYP11A, 2 key enzymes for progesterone synthesis. In conclusion, Notch signaling plays important role in regulating progesterone secretion in murine luteal cells.//////////////////
A link between Notch and progesterone maintains the functionality of the rat corpus luteum. [Accialini P et al. (2014) In this study, we investigated the interaction between the Notch pathway and progesterone to maintain the functionality of the corpus luteum (CL). When Notch signaling is activated, the γ-secretase complex releases the active intracellular domains (NICD) of their receptors, which exert survival effects. We designed studies to analyze whether the in vitro inhibition of Notch affects progesterone production, steroidogenic regulators, apoptotic parameters, and signaling transduction pathways in the cultures of CL isolated from pregnant and superovulated rats. We detected a decrease in progesterone production when corpora lutea (CL) were incubated with N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor. This effect could be in part due to the decrease detected in the CL protein levels of P450scc because STAR and 3β-hydroxysteroid dehydrogenase were not affected by Notch inhibition. Besides, the addition of aminoglutethimide to the CL culture medium decreased NICD of NOTCH1. We observed an increase in the expression of active CASPASE3 (CASP3) after inhibition by Notch, which was reversed by the presence of progesterone. The BAX:BCLXL ratio was increased in CL treated with DAPT and the presence of progesterone reversed this effect. In addition, phosphorylation of AKT was inhibited in CL treated with DAPT, but had no effect on ERK activation. To demonstrate that the action of DAPT is specifically related with the inhibition of Notch, CLs were incubated with DLL4 antibody and a decrease in progesterone production was detected. These results suggest the existence of a novel link between progesterone and the Notch signaling pathway to maintain the functionality of the CL.//////////////////
Primordial follicle assembly was regulated by notch signaling pathway in the mice. Chen CL 2014 et al.
Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P<0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P<0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.
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Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse. Feng YM 2014 et al.
A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.
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P4 down-regulates Jagged2 and Notch1 expression during primordial folliculogenesis. Guo M et al. Nest breakdown and primordial folliculogenesis of the mouse ovary can be inhibited by progesterone (P4) and Notch signaling inhibitors. However, the relationship between these two signals during this process remains unknown. In the present study, transcript levels of Jagged2, Notch1, and their target, Hey2, increased markedly in ovaries during the beginning stage of folliculogenesis (17.5 days post coitus (dpc) to birth). Maternal P4 levels decreased simultaneously. We found that maternal midpregnancy P4 levels significantly inhibited Jagged2, Notch1, and Hey2 expression, and follicle formation in vitro. Maintaining high maternal P4 levels by daily injection also significantly suppressed the expression of Jagged2, Notch1, and Hey2, and follicle formation during late pregnancy. Based on immunohistochemistry, Jagged2 was localized in oocytes and Notch1 was strongly stained in pre-granulosa cells in 19.5 dpc ovaries. Suppression of their function by antibody addition and RNAi markedly inhibited nest breakdown and follicle formation. Taken together, these results demonstrate that maternal P4 levels during midpregnancy can inhibit the expression of Jagged2 and Notch1, which are involved in primordial folliculogenesis, in the mouse fetal ovary.
Notch Signaling Is Involved in Ovarian Follicle Development by Regulating Granulosa Cell Proliferation. Zhang CP et al. Notch signaling is an evolutionarily conserved pathway, which regulates cell proliferation, differentiation, and apoptosis. It has been reported that the members of Notch signaling are expressed in mammalian ovaries, but the exact functions of this pathway in follicle development is still unclear. In this study, primary follicles were cultured in vitro and treated with Notch signaling inhibitors, L-658,458 and N-N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). We found that the cultured follicles completely stopped developing after L-658,458 and DAPT treatment, most of the granulosa cells were detached, and the oocytes were also degenerated with condensed cytoplasma. Further studies demonstrated that the proliferation of granulosa cells was dependent on the Notch signaling. L-658,458 and DAPT treatment inhibited proliferation of in vitro cultured primary granulosa cells and decreased the expression of c-Myc. Lentivirus mediated overexpression of Notch intracellular domain 2, and c-Myc could promote the proliferation of granulosa cells and rescue the growth inhibition induced by L-658,458 and DAPT. In conclusion, Notch signaling is involved in follicular development by regulating granulosa cell proliferation.
Notch signaling controls germline stem cell niche formation in the Drosophila ovary. [Song X et al. Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, we show that Notch signaling controls formation and maintenance of the GSC niche and that cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. Our results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function.
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Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4). Jovanovic VP et al. BACKGROUND: In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary. METHODS: Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (alpha-SMA) detection. RESULTS: We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis. CONCLUSIONS: Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated.
////Role of the DLL4-NOTCH System in PGF2alpha-Induced Luteolysis in the Pregnant Rat. Hernandez F et al. We investigated the expression and cell localization of NOTCH1, NOTCH4 and the ligand DlLL4 in corpus luteum (CL) from pregnant rats during PGF2alpha-induced luteolysis. We also examined serum progesterone (P(4)) and CL proteins related to apoptosis after local administration of the notch inhibitor DAPT. Specific staining for NOTCH1 and 4 receptors was detected predominantly in large and small luteal cells. Furthermore, the staining was evident in the nuclei of luteal cells in coincidence with the fact that the notch intracellular domain is translocated to the nucleus, where it regulates gene expression. Additionally, we detected diffuse cytoplasmic immunostaining for DLL4 in small and large luteal cells, in accordance with the information that DLL4 undergoes proteolytic degradation after receptor binding. The mRNA expression of Notch1, Notch4 and Dll4 in CL isolated on Day 19 of pregnancy decreased significantly after the administration of PGF2alpha. Consistent with the mRNA results, administration of PGF2alpha to pregnant rats on Day 19 of pregnancy decreased the protein fragment corresponding to the cleaved forms of NOTCH1/4 CL receptors. In contrast, no significant changes were detected in protein levels for the ligand DLL4. The local intrabursal administration of DAPT decreased serum P(4) levels, increased luteal levels of active CASP3 and the BAX/BCL2 ratio 24 h after the treatment. These results support a luteotropic role for notch signaling to promote luteal cell viability and steroidogenesis, and suggest that the luteolytic hormone PGF2alpha may act in part by reducing the expression of some notch system members.
This gene was found in a mouse DNA array analysis of transcripts expressed in mouse preovulatory follicles.
Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation Vorontchikhina MA, et al .
Notch signaling functions to regulate cell-fate decisions by modulating differentiation, proliferation, and survival of cells. Notch receptors and ligands are expressed in embryonic vasculature and are required for the remodeling of the primary embryonic vasculature of mice. Here, we characterize the expression patterns of Notch1, Notch4, and Jagged1 proteins during the process of folliculogenesis and corpus luteum formation in the mouse ovary, an organ with dynamic physiological angiogenic growth. These Notch proteins and ligand are expressed in a subset of ovarian vessels, including both mature ovarian vasculature as well as angiogenic neovessels. Their expression in the ovary was found in both endothelial and vascular associated mural cells. Our data suggest a complex regulatory role for the Notch signaling pathway during mouse oogenesis and ovarian neovascularization.
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Mutations |
1 mutations
Species: mouse
Mutation name:
type: targeted overexpression
fertility: infertile - ovarian defect
Comment: Constitutive Notch Signaling Causes Abnormal Development of the Oviducts, Abnormal Angiogenesis, and Cyst Formation in Mouse Female Reproductive Tract. Ferguson L et al. (2016) The Notch signaling pathway is critical for the differentiation of many tissues and organs in the embryo. To study the consequences of Notch1 gain-of-function signaling on female reproductive tract development, we used a cre-loxP strategy and Amhr2-cre transgene to generate mice with conditionally activated Notch1 (Rosa(Notch1)). The Amhr2-cre transgene is expressed in the mesenchyme of developing female reproductive tract and in granulosa cells in the ovary. Double transgenic Amhr2-cre, Rosa(Notch1) females were infertile, whereas control Rosa(Notch1) mice had normal fertility. All female reproductive organs in mutants showed hemorrhaging of blood vessels progressing with age. The mutant oviducts did not develop coiling, and were instead, looped around the ovary. There were multiple blockages in the lumen along the oviduct length, creating a barrier for sperm or oocyte passage. Mutant females demonstrated inflamed uteri with increased vascularization and an influx of inflammatory cells. Additionally, older females developed ovarian, oviductal, and uterine cysts. The significant change in gene expression was detected in the mutant oviduct expression of Wnt4, essential for female reproductive tract development. Similar oviductal phenotypes have been detected previously in mice with activated Smo and in beta-catenin, Wnt4, Wnt7a and Dicer conditional knockouts, indicating a common regulatory pathway disrupted by these genetic abnormalities.//////////////////
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