| KIT ligand | OKDB#: 165 |
| Symbols: | KITLG | Species: | human | ||
| Synonyms: | SF, MGF, SCF, SLF, DCUA, FPH2, FPHH, KL-1, Kitl, SHEP7, DFNA69 | Locus: | 12q21.32 in Homo sapiens |
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| General Comment |
Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice. Zhang H et al. (2014) The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals.//////////////////
Stem cell factor is a hematopoietic growth factor and ligand for the KIT tyrosine kinase receptor. In the mouse, this growth factor is encoded by Sl ('steel'), a gene critical to the development of several distinct cell lineages during embryonic life as well as having important effects on hematopoiesis in the adult animal. Using probes based on the sequence of rat stem cell factor (SCF), Martin et al. (1990) isolated cDNA and genomic clones of human SCF. On the basis of analysis of somatic cell hybrids between human cells and either hamster or mouse cells, Geissler et al. (1991) reported that the human gene maps to 12q14.3-qter. Mathew et al. (1992)
regionalized the MGF gene to 12q22 by in situ hybridization.
//////Cellular and molecular regulation of the activation of mammalian primordial follicles: somatic cells initiate follicle activation in adulthood. Zhang H et al. (2015) The first small follicles to appear in the mammalian ovaries are primordial follicles. The initial pool of primordial follicles serves as the source of developing follicles and oocytes for the entire reproductive lifespan of the animal. Although the selective activation of primordial follicles is critical for female fertility, its underlying mechanisms have remained poorly understood. A search of PubMed was conducted to identify peer-reviewed literature pertinent to the study of mammalian primordial follicle activation, especially recent reports of the role of primordial follicle granulosa cells (pfGCs) in regulating this process. In recent years, molecular mechanisms that regulate the activation of primordial follicles have been elucidated, mostly through the use of genetically modified mouse models. Several molecules and pathways operating in both the somatic pfGCs and oocytes, such as the phosphatidylinositol 3 kinase (PI3K) and the mechanistic target of rapamycin complex 1 (mTORC1) pathways, have been shown to be important for primordial follicle activation. More importantly, recent studies have provided an updated view of how exactly signaling pathways in pfGCs and in oocytes, such as the KIT ligand (KL) and KIT, coordinate in adult ovaries so that the activation of primordial follicles is achieved. In this review, we have provided an updated picture of how mammalian primordial follicles are activated. The functional roles of pfGCs in governing the activation of primordial follicles in adulthood are highlighted. The in-depth understanding of the cellular and molecular mechanisms of primordial follicle activation will hopefully lead to more treatments of female infertility, and the current progress indicates that the use of existing primordial follicles as a source for obtaining fertilizable oocytes as a new treatment for female infertility is just around the corner.//////////////////
NCBI Summary: This gene encodes the ligand of the tyrosine-kinase receptor encoded by the KIT locus. This ligand is a pleiotropic factor that acts in utero in germ cell and neural cell development, and hematopoiesis, all believed to reflect a role in cell migration. In adults, it functions pleiotropically, while mostly noted for its continued requirement in hematopoiesis. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] |
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| General function | Ligand, Growth factor, Cell adhesion molecule | ||||
| Comment | Stem cell factor (KL) augmented the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures (Martin et al., 1990). The receptor encoded by the W (c-kit) locus is expressed on the membrane of mouse primordial germ cells, whereas stem cell factor (SCF), encoded by the Sl (Steel) locus, is expressed on the membrane of somatic cells associated with both the primordial germ cell migratory pathways and homing sites. Using an in vitro short time assay which allows a quantitative measure of adhesion between cells it was shown, that SCF/c-kit interaction can modulate primordial germ cell adhesion to somatic cells (Pesce et al., 1997). | ||||
| Cellular localization | Secreted, Plasma membrane | ||||
| Comment | KL exists in both cell surface and soluble forms. The soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner (Huang et al., 1992). | ||||
| Ovarian function |
Follicle development, Initiation of primordial follicle growth, Primary follicle growth, Preantral follicle growth, Germ cell development, Germ cell migration, Oogenesis, Oocyte growth, Oocyte maturation
, First polar body extrusion |
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| Comment |
Increased SCF in Follicular Fluid and Granulosa Cells Positively Correlates With Oocyte Maturation, Fertilization, and Embryo Quality in Humans. Tan J et al. (2017) Stem cell factor (SCF), which is derived from granulosa cells (GCs), plays a key role in the process of follicular development and oocyte maturation. The present study aimed to explore whether the levels of SCF in follicular fluid (FF) and GCs can be used as a potential marker for predicting oocyte developmental potential. Follicular fluid and GC samples from 150 female patients undergoing intracytoplasmic sperm injection were collected in this study. The SCF concentrations in FFs and SCF messenger RNA (mRNA) in GCs were evaluated by using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. The results showed that the levels of SCF protein and mRNA were significantly associated with oocyte maturation, normal fertilization, cleavage, and embryo quality. Moreover, the levels of SCF protein and mRNA in pregnancy group were also higher than those in the nonpregnancy group. The cutoff value of SCF in FF for predicting high-quality embryo was 1.346, with a sensitivity of 57.8% and a specificity of 72.4%, and the cutoff value of SCF in GCs for predicting high-quality embryo was 6.650, with a sensitivity of 64.4% and a specificity of 78.1%. In conclusion, our results showed a positive and statistically significant relationship between SCF level and oocyte maturation, normal fertilization, cleavage, embryo quality, and clinical pregnancy. Therefore, the levels of SCF in FF and GCs might be considered as a new marker for predicting oocyte developmental potential.//////////////////
MAPK3/1 Participates in the Activation of Primordial Follicles through mTORC1-KITL Signaling. Zhao Y et al. (2017) The majority of ovarian primordial follicles are preserved in a dormant state to maintain the female reproductive lifespan, and only a few primordial follicles are activated to enter the growing follicle pool in each wave. Recent studies have shown that primordial follicular activation depends on mammalian target of rapamycin complex 1 (mTORC1)-KIT ligand (KITL) signaling in pre-granulosa cells and its receptor (KIT)-phosphoinositol 3 kinase (PI3K) signaling in oocytes. However, the upstream regulator of mTORC1 signaling is unclear. The results of the present study showed that the phosphorylated mitogen-activated protein kinase3/1 (MAPK3/1) protein is expressed in some primordial follicles and all growing follicles. Culture of 3 days post-parturition (dpp) ovaries with the MAPK3/1 signaling inhibitor U0126 significantly reduced the number of activated follicles and was accompanied by dramatically reduced granulosa cell proliferation and increased oocyte apoptosis. Western blot and immunofluorescence analyses showed that U0126 significantly decreased the phosphorylation levels of Tsc2, S6K1, and rpS6 and the expression of KITL, indicating that U0126 inhibits mTORC1-KITL signaling. Furthermore, U0126 decreased the phosphorylation levels of Akt, resulting in a decreased number of oocytes with Foxo3 nuclear export. To further investigate MAPK3/1 signaling in primordial follicle activation, we used phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibitor bpV(HOpic) to promote primordial follicle activation. In this model, U0126 also inhibited the activation of primordial follicles and mTORC1 signaling. Thus, these results suggest that MAPK3/1 participates in primordial follicle activation through mTORC1-KITL signaling. This article is protected by copyright. All rights reserved.//////////////////
Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary. Ezzati MM et al. (2015) Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37 °C and 5 % CO2 for 60-90 min with (n = 3) or without (n = 3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p = 0.007 in paired samples and p = 0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p = 0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation.//////////////////
Fertility: the role of mTOR signaling and KIT ligand. Hsueh AJ et al. Activation of a limited pool of diminishing ovarian follicles determines women's reproductive lifespan. A recent rodent study describes the role of mTOR signaling and KIT ligand in granulosa cells of primordial follicles for follicle activation and for reproductive lifespan regulation.
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Kit ligand and glial-derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro. Esmaielzadeh F et al. In vitro growth of preantral follicles has the potential to produce considerable numbers of competent oocytes for use in medicine, agriculture, and even wildlife conservation. The critical regulatory role of growth factors and hormones in the development of preantral follicles has been established. This study investigated the effect of glial-derived neurotropic factor (GDNF) and kit ligand (KL) on the in vitro development of ovine preantral follicles. Results indicated that both GDNF and KL significantly improved activation of primordial follicles, similar to co-addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are commonly used for in vitro follicular development. Importantly, GDNF had a more profound effect on follicle health, development, and differentiation compared with KL alone. Furthermore, the combination of GDNF and KL in the presence of EGF and bFGF had a positive, synergic effect on health, development, and differentiation of preantral follicles, as determined by histological and hormonal assessments. The results of this study may provide a foundation for further studies that will unravel the molecular mechanisms of follicular development to further improve the current status of in vitro preantral follicle culture. Mol. Reprod. Dev. ? 2012 Wiley Periodicals, Inc.
Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro. Celestino JJ et al. The aims of this study were to investigate steady-state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT-PCR was used to analyze caprine steady-state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1-3 mm) and large (3-6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM(+)) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT-PCR demonstrated an increase in steady-state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady-state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM(+) after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth. Mol. Reprod. Dev. 2009. (c) 2009 Wiley-Liss, Inc.
Kit ligand promotes first polar body extrusion of mouse preovulatory oocytes. Ye Y et al. ABSTRACT: BACKGROUND: Shortly after stimulation by the preovulatory surge of luteinizing hormone (LH), oocytes arrested at the late prophase I resume meiosis characterized by germinal vesicle breakdown (GVBD), chromosome condensation, and extrusion of the first polar body in preparation for fertilization and early embryonic development. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. To identify candidate ligand-receptor pairs potentially involved in the process of oocyte maturation, we performed DNA microarray analyses of ovarian transcripts in mice and identified Kit ligand (Kitl) as an ovarian factor stimulated by the LH/hCG surge. The purpose of this study is to investigate the roles of KITL in the nuclear and cytoplasmic maturation of preovulatory mouse oocytes. METHODS: The levels of Kitl and c-kit transcripts in mouse ovaries and isolated ovarian cells were determined by real-time RT-PCR, while expression of KITL protein was examined by immunohistochemistry. Follicle culture, cumulus-oocyte complexes (COC) and denuded oocytes culture were used to evaluate the effect of KITL on mouse oocyte nuclear maturation. To assess the effect of KITL treatment on the cytoplasmic maturation of preovulatory oocytes, we performed in vitro maturation of oocytes followed by in vitro fertilization. RESULTS: Major increase of Kitl transcripts in granulosa cells and mouse ovaries, and predominant expression of c-kit in preovulatory oocytes were identified by real-time RT-PCR. Predominant expression of KITL protein was found in granulosa cells of preovulatory and small antral follicles at 4 h after hCG treatment. In vitro cultures demonstrated that treatment with KITL enhanced first polar body extrusion in a dose-dependent manner. Moreover, treatment of COC with KITL enhanced first polar body extrusion with increase in cyclin B1 synthesis which is important for the progression of meiotic maturation after GVBD. In contrast, treatment of cultured preovulatory follicles with KITL did not affect GVBD and KITL has no effect on cytoplasmic maturation of preovulatory oocytes. CONCLUSIONS: Our findings suggest potential paracrine roles of KITL in the nuclear maturation of preovulatory oocytes by promoting first polar body extrusion.
Godin et al. (1991) demonstrate the control of the early germ-line population by SCF, which is an essential requirement for primordial germ cell survival. The addition of KL to growing follicles cultured in collagen gels resulted in a 67% increase in the rate of oocyte growth (Packer et al., 1994). Motro and Bernstein (1993) showed that the cessation of murine oocyte growth, at the transition of the follicle to the antral stage, is associated with the
cessation of Steel expression in the cumulus granulosa cells in the vicinity of the oocyte. These observations suggest a role for the Kit signaling pathway in oocyte growth or in meiotic arrest. The presence of KL resulted in a significant, albeit transient, delay in the progression of spontaneous meiotic maturation in the rat, using the indices of germinal vesicle breakdown and polar body formation (Ismail et al., 1996). After 14 days of KL treatment, sections from rat ovaries contained 17% primordial follicles (stage 0) and 83% developing follicles (stage 1-4) per section demonstrating a dramatic induction of primordial follicle development by KL (Parrott and Skinner, 1999). Parrott and Skinner (1997 and 1998) demonstrate that KL can directly stimulate theca cell growth and steroid production during bovine follicular development. Furthermore, granulosa cell-derived KL stimulated keratinocyte growth factor and hepatocyte growth factor. Furthermore, in bovine organ culture KL significantly increased the number of theca cell layers around primary follicles. Experiments using purified stromal interstitial cell cultures showed that KL stimulated ovarian stromal cell proliferation in a dose-dependent manner. Stromal cell differentiation into theca cells was analyzed by the induction of theca cell functional markers (i.e., androstenedione and progesterone production) expression in bovine thecal cells (Parrott and Skinner, 2000). Reddy P,et al reported the activation of Akt (PKB) and suppression of FKHRL1 in mouse and rat oocytes by stem cell factor during follicular activation and development. Although communications between mammalian oocytes and their surrounding granulosa cells mediated by the Kit-Kit ligand (KL, or stem cell factor, SCF) system have been proven to be crucial for follicular development, Kit downstream signaling pathways in mammalian oocytes are largely unknown. In this study, by using ovaries and isolated oocytes from postnatal mice and rats, we demonstrated for the first time that components of the PI3 kinase pathway, the serine/threonine kinase Akt (PKB) which enhances cellular proliferation and survival, and an Akt substrate FKHRL1 which is a transcription factor that leads to apoptosis and cell cycle arrest, are expressed in mammalian oocytes. By using an in vitro oocytes culture system, we found that oocytes-derived Akt and FKHRL1 are regulated by SCF. Treatment of cultured oocytes with SCF cannot only rapidly phosphorylate and activate Akt, but also simultaneously phosphorylate and may therefore functionally suppress FKHRL1, through the action of PI3 kinase. Together with our in situ hybridization and immunohistochemistry data that Akt and FKHRL1 are mostly expressed in oocytes in primordial and primary ovaries and reports that FKHRL1 gene-deficient mice exhibited excessive activation from primordial to primary follicles as well as enlarged oocyte sizes, we suppose that in mammalian oocytes, actions of granulosa cell derived SCF on primordial to primary follicle transition and subsequent follicle development may involve activation of Akt and inhibition of FKHRL1 activities in oocytes. The role of oocyte's Akt may be to enhance follicle development and the role of oocyte's FKHRL1 may be to inhibit follicle development. We propose that the cascade from granulosa cell SCF to oocyte Kit-PI3 kinase-Akt-FKHRL1 may play an important role to regulate the growth rate of mammalian oocytes and hypothetically also the oocyte secretion of factors that may regulate the activation and early development of ovarian follicles. Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture. Carlsson IB et al. The receptor tyrosine c-Kit and its cognate ligand, c-Kit ligand (KL, stem cell factor, SCF), are involved in ovarian follicular development in several animal species. We studied the expression of KL and c-Kit using in situ hybridization and immunohistochemistry in donated human ovarian cortical tissue. The KL transcripts were expressed in granulosa cells of primary follicles, whereas the expression of c-Kit was confined to the oocyte and granulosa cells in primary and secondary follicles. We employed an ovarian organ culture using firstly serum-containing and then serum-free medium to study the effects of KL and an anti-c-Kit antibody, ACK2, on the development and survival of ovarian follicles in vitro. Culture of ovarian cortical slices for 7 days resulted in a 37% increase in the number of primary follicles and a 6% increase in secondary follicles. The proportion of viable follicles decreased in all cultures. The addition of KL (1, 10 and 100 ng/ml) into the culture media did not affect the developmental stages of the follicles or the proportion of atretic follicles. Inclusion of ACK2 (800 ng/ml) in the culture medium significantly increased the proportion of atretic follicles on days 7 (49 vs 28% in control cultures) and 14 (62 vs 38%) of culture. In conclusion, c-Kit and KL are expressed in human ovaries during follicular development. Blocking the c-Kit receptor induces follicular atresia. The KL/c-Kit signaling system is likely to control the survival of human ovarian follicles during early follicular development. Phosphorylation and inactivation of glycogen synthase kinase-3 by soluble kit ligand in mouse oocytes during early follicular development. Liu L et al. Communication between mammalian oocytes and their surrounding granulosa cells through the Kit-Kit ligand (KL, or stem cell factor, SCF) system has been shown to be crucial for follicular development. Our previous studies (Reddy et al. 2005, Liu et al. 2006) have indicated that the intra-oocyte KL-Kit-PI3 kinase (PI3K)-Akt-Foxo3a cascade may play an important role in follicular activation and early development. In the present study, using in situ hybridization and in vitro culture of growing oocytes from 8-day-old postnatal mice, we have demonstrated that another Akt substrate, glycogen synthase kinase-3 (GSK-3), is expressed in growing oocytes. Also, treatment of cultured mouse oocytes with soluble KL not only leads to increased Akt kinase activity in the oocytes, which can phosphorylate recombinant GSK-3 in vitro, but also leads to phosphorylation of oocyte GSK-3alpha and GSK-3beta, which can result in the inactivation of GSK-3 function in oocytes. In addition, we have shown that the regulation of GSK-3alpha and GSK-3beta in cultured oocytes by soluble KL is accomplished through PI3K, since the PI3K-specific inhibitor LY294002 completely abolished the KL-induced phosphorylation of GSK-3alpha and GSK-3beta. Moreover, blockage of the Kit signaling pathway by a Kit function-blocking antibody, ACK2, resulted in reduced phosphorylation of GSK-3. Taken together, our data suggest that the cascade from granulosa cell-derived KL to Kit-PI3K-Akt-GSK-3 in oocytes may take part in regulation of oocyte growth and early ovarian follicular development. Kit Ligand 2 Promotes Murine Oocyte Growth In Vitro. Thomas FH et al. Oocyte-granulosa cell communication, mediated by paracrine factors, is essential for oocyte development. Kit ligand (KITL) is expressed in granulosa cells as soluble (KITL1) or membrane-associated (KITL2) proteins. However, the relative biopotency of each isoform during oocyte development is unknown. Our initial results showed that Kitl2 was down-regulated in cultured granulosa cells. To determine the effect of the two isoforms of KITL on oocyte growth, Kitl-deficient fibroblasts were transfected with constructs expressing either KITL1 or KITL2 and growing oocytes were isolated from 12-day-old mice and cultured on the transfected fibroblasts for 2 days. At the end of culture, oocyte diameters were measured, the incidence of spontaneous germinal vesicle breakdown (GVBD) was noted, and oocytes were analyzed for KIT receptor expression. Oocyte growth occurred only in the presence of the KITL2-producing fibroblasts, and suppression of KITL2 expression impaired oocyte growth. Up-regulation of KIT expression occurred in the presence of KITL2, but not KITL1. The presence of KITL2 inhibited spontaneous GVBD. Meiosis inhibitors did not attenuate the GVBD that occurred in the absence of KITL2, suggesting that this process reflects oocyte degeneration rather than meiotic progression. These results indicate that KITL2 is the principal KITL isoform required for oocyte growth and survival in vitro. KIT-KIT Ligand in the Growth of Porcine Oocytes in Primordial Follicles. Moniruzzaman M et al. Mammalian ovaries are endowed with a huge number of small oocytes in primordial follicles (primordial oocytes). The mechanism regulating initiation of oocyte growth and follicular development is not well understood. Several growth factors and cytokines are known to be involved in oocyte growth and follicular development. Herein, the involvement of KIT, a receptor tyrosine kinase, and its ligand, KIT ligand (KL), in the initiation of porcine oocyte growth was examined. At first, KIT expression was examined immunohistochemically in primordial oocytes from neonatal (10-20 days) and prepubertal (about 6 months) pigs. Similar expression of KIT was detected in all oocytes from both the neonatal and prepubertal pigs. Next, to examine the growth of primordial oocytes, ovarian tissues containing primordial oocytes were xenotransplanted into immunodeficient SCID mice. Primordial oocytes from the neonatal pigs grew with follicular development as described previously, whereas those from the prepubertal pigs did not initiate growth in the xenografts after 2 months. To stimulate the growth of primordial oocytes from the prepubertal pigs, they were cultured in a medium supplemented with KL (50 and 100 ng/ml) for 1 or 3 days before xenografting. After 2 months, however, the oocytes did not grow, and the primordial follicles did not develop, although a higher number of primordial oocytes survived in the KL-treated tissues. These results suggest that KIT-KL might not be associated with the growth initiation of porcine primordial oocytes, although they do enhance the survival of the oocytes. |
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| Expression regulated by | FSH, LH, Growth Factors/ cytokines, AMH, GDF9, PMSG, TGFbeta, activin, IGF1 | ||||
| Comment | Anti-Müllerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB. Fu YX et al. (2020) Anti-Müllerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318-321 bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.//////////////////Antim?an hormone regulates stem cell factor expression in human granulosa cells. Hu R 2014 et al. OBJECTIVE To determine whether there is a correlation between antim?an hormone (AMH) and stem cell factor (SCF) in serum, follicular fluid (FF), and granulosa cells (GCs), and to investigate a possible regulatory mechanism of AMH on SCF in human granulosa cells. DESIGN Prospective clinical and experimental study. SETTING Academic center. PATIENT(S) 163 women undergoing IVF. INTERVENTION(S) Serum, FF, and GCs obtained in all women, primary cultures of human GCs. MAIN OUTCOME MEASURE(S) AMH and SCF were analyzed in serum, FF, and GCs, using enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and immunoblotting. RESULT(S) There was a significant negative correlation between AMH and SCF protein level in FF, and in the mRNA expression of AMH and SCF in GCs. Conversely, there was no correlation between AMH and SCF levels in serum. In primary cultures of human GCs, SCF was down-regulated by treatment with recombinant human AMH and was increased by cyclic adenosine 3':5' monophosphate (cAMP) in a dose-dependent manner. A protein kinase A (PKA) inhibitor (H89) significantly reversed the effects of recombinant human AMH and cAMP on SCF mRNA and protein expression. CONCLUSION(S) This is the first report on a modulatory role for AMH as an ovarian/follicular autocrine/paracrine factor controlling SCF expression via the cAMP/PKA pathway. ///////////////////////// Mouse GDF9 decreases KITL gene expression in human granulosa cells. Tuck AR 2014 et al. Kit ligand (KITL) is an important granulosa cell-derived growth factor in ovarian folliculogenesis, but its expression and function in human granulosa cells are currently poorly understood. Based on studies performed in animal models, it was hypothesised that KITL gene expression in human granulosa cells is regulated by androgens and/or growth differentiation factor 9 (GDF9). We utilised two models of human granulosa cells, the KGN granulosa tumour cell line and cumulus granulosa cells obtained from preovulatory follicles of women undergoing assisted reproduction. Cells were treated with combinations of 5a-dihydrotestosterone (DHT), recombinant mouse GDF9, and the ALK4/5/7 inhibitor SB431542. KITL mRNA levels were measured by quantitative real-time PCR. No change in KITL mRNA expression was observed after DHT treatment under any experimental conditions, but GDF9 treatment resulted in a significant decrease in KITL mRNA levels in both KGN and cumulus cells. The effect of GDF9 was abolished by the addition of SB431542. These results indicate that KITL is not directly regulated by androgen signalling in human granulosa cells. Moreover, this study provides the first evidence that GDF9 negatively regulates KITL gene expression in human granulosa cells providing new information on the regulation of these important growth factors in the human ovary. ///////////////////////// Activin A selectively represses expression of the membrane-bound isoform of Kit ligand in human fetal ovary. Childs AJ et al. Germ cell-expressed activin A promotes germ cell survival and proliferation indirectly, possibly by maintaining ovarian pregranulosa cells in an undifferentiated state and preventing primordial follicle formation until oocytes have matured sufficiently. We have previously demonstrated that recombinant activin A can repress the expression of Kit ligand (KITLG) in cultures of human fetal ovaries, and here we extend this finding to show that this repression acts selectively on the membrane-bound isoform of Kit ligand without affecting the soluble form, providing a potential mechanism for suppressing direct interaction between somatic and germ cells and thus inhibiting precocious primordial follicle formation. Cyclic secretion of luteinizing hormone results in elevated Steel expression in mural granulosa cells and decreased levels of c-kit transcripts in stromal-derived cells. PMSG-treated rats had 3.5-fold greater abundance of KL mRNA compared to untreated rats. The abundance of KL mRNA further increased to 7-fold over control levels at 6 hr after PMSG-primed rats were treated with human chorionic gonadotropin (hCG). By contrast, treatment of rats with diethylstilbestrol to stimulate follicular growth did not cause any change in the abundance of KL transcripts (Ismail et al., 1996). Treatment of bovine granulosa cells with FSH and hCG directly stimulated KL expression and (Parrott and Skinner 1998) TGF-beta strongly inhibited dibutyryl cAMP-induced KL expression in ovarian suface epithelial cells (Ismail et al., 1999). The results of Joyce et al. (1999) show, that oocytes regulate granulosa cell kit ligand mRNA levels in a way that is characteristic of the stage of growth and development of the oocyte. Differential Regulation of Kit Ligand A Expression in the Ovary by IGF-I via Different Pathways. Yao K 2013 et al. Kit ligand (KITL) plays indispensable roles both in primordial follicle activation and in the maintenance of meiotic arrest of the oocyte. The regulation of KITL expression in the ovary, however, remains largely unknown. In the zebrafish, there are 2 paralogues of KITL, kitlga and kitlgb, and 2 Kit receptors, kita and kitb. Consistent with the situation in mammals, kitlga is only expressed in the ovarian follicle cells, and its cognate receptor kita is expressed in the oocyte. In the present study, we demonstrated that the expression of kitlga was promoted by IGF-I through its receptor IGF-IR. The stimulation involved transcription but not translation, suggesting that the kitlga gene is likely a direct downstream target of IGF-I signaling. Further experiments showed that the stimulatory effect of IGF-I was mediated by phosphatidyl inositol 3-kinase-Akt pathway. IGF-I also activated MEI-ERK pathway; however, this pathway suppressed kitlga expression. The regulation of kitlga expression by IGF-I appeared to depend on the stage of follicle development with a greater induction at early stage than late stage. This may be related to changes in IGF-I signaling pathways and/or local paracrine environment. In support of this were the differential expression of IGF-I receptors (igf1ra and igf1rb) and responsiveness of IGF-I signaling pathways, especially the phosphatidyl inositol 3-kinase-Akt pathway. Furthermore, the IGF-I-induced kitlga expression was inhibited by epidermal growth factor, an oocyte-derived paracrine factor in the zebrafish follicle. This study provides evidence for a controlling mechanism underlying the regulation of KITL expression in the ovary. ///////////////////////// Analysis of Hypomorphic KitlSl Mutants Suggests Different Requirements for KITL in Proliferation and Migration of Mouse Primordial Germ Cells Mahakali Zama A, et al . Germ cell development in mice is initiated when a small number of primordial germ cells (PGCs) are set aside from somatic cells during gastrulation. In the subsequent 4 to 5 days, PGCs enter the hindgut, undergo a directed migration away from the hindgut into the developing gonads, and undergo a massive increase in cell number. It is well established that Kit ligand (KITL, also known as stem cell factor and mast cell growth factor) is required for the survival and proliferation of PGCs. However, there is little information on a direct role for KITL in PGC migration. By comparing the effects of multiple Kitl mutations, including two N-ethyl-N-nitrosourea-induced hypomorphic mutations, we were able to distinguish stages of PGC development that are preferentially affected by certain mutations. We provide evidence that the requirements for KITL in proliferation are different in PGCs before and after they start migrating, and different levels of KITL function are required to support PGC proliferation and migration. This study illustrates the usefulness of an allelic series of mutations to dissect developmental processes and suggests that these mutants may be useful for further studies of molecular mechanisms of KITL functions in gametogenesis. KIT/KIT Ligand in Mammalian Oogenesis and Folliculogenesis: Roles in Rabbit and Murine Ovarian Follicle Activation and Oocyte Growth. Hutt KJ et al. In rodent ovaries KIT ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth and follicle survival. Studies were undertaken to determine if KITL and KIT carry out similar activities in rabbits. Kitl and Kit mRNA and protein were localised to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary and growing primary follicles increased significantly in recombinant mouse KITL treated explants compared to untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse and these effects were inhibited by co-culture with KITL neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiological roles of KITL and KIT may differ between species and this has important implications for the design of in vitro culture systems for mammalian folliculogenesis, including the human. | ||||
| Ovarian localization | Primordial Germ Cell, Cumulus, Granulosa, Surface epithelium | ||||
| Comment | In situ hybridization studies showed a gradient in signal intensity in rat antral follicles, with greatest intensity observed in the cumulus granulosa cells enclosing the oocyte, and less signal occurring in the granulosa cells furthest from the oocyte (Ismail et al., 1996). KL mRNA and protein were detected in the ovarian surface epithelial cell layer of rat ovaries (Ismail et al., 1999). Changes in mouse granulosa cell gene expression during early luteinization. McRae RS et al. Changes in gene expression during granulosa cell luteinization have been measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with pregnant mare serum gonadotropin (PMSG) or PMSG followed, 48 h later, by human chorionic gonadotropin (hCG). Granulosa cells were collected from preovulatory follicles after PMSG injection or PMSG/hCG injection and SAGE libraries generated from the isolated mRNA. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by hCG and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in steroidogenesis as well as clusters of genes involved in modeling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signaling. The SAGE libraries described here provide a base for functional investigation of the regulation of granulosa cell luteinization. | ||||
| Follicle stages | Primordial, Primary, Secondary, Antral, Preovulatory, Corpus luteum | ||||
| Comment | In fetal and adult ovine ovaries, SCF mRNA was detected in the granulosa cells of follicles at all stages of follicle growth (primordial through antral). In fetal ovaries at day 90 of gestation, SCF was expressed in the subepithelial mesenchymal cells of the ovarian cortex (Tisdall et al., 1997). In ovine, SCF mRNA was localized within Day 3 and Day 10 CL by in situ hybridization and was expressed throughout luteal tissue on both days examined (Gentry et al., 1996). | ||||
| Phenotypes | |||||
| Mutations |
6 mutations
Species: ovine
Species: mouse
Species: mouse
Species: mouse
Species: mouse
Species: mouse
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| Genomic Region | show genomic region | ||||
| Phenotypes and GWAS | show phenotypes and GWAS | ||||
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| last update: | Feb. 17, 2020, 2:31 p.m. | by: | hsueh email: |
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