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Nodal regulates ovarian functions in zebrafish. Zayed Y et al. (2020) Nodal, a member of the transforming growth factor-β (TGF-β) superfamily, plays critical roles during embryo development. Several studies suggest that Nodal also regulates reproduction. The objective of this study was to investigate if Nodal is expressed in zebrafish ovary and if it is involved in the regulation of ovarian functions. Using real-time PCR, we detected two Nodal homologs, nodal-related (ndr)1, and ndr2 in zebrafish ovarian follicles. We further compared the mRNA levels of ndr1, ndr2, and their receptors between maturational incompetent early vitellogenic follicles (stage IIIa) and mid-to late-vitellogenic follicles (stage IIIb) which are capable of undergoing maturation when they are induced by hormones. We found that mRNAs for ndr1 and ndr2, as well as two type I receptors, acvr1ba, and acvr1c, were significantly increased in follicular cells isolated from stage IIIb follicles. In primary cultures of ovarian follicular cells, treatment with recombinant human Nodal inhibited cell proliferation. On the other hand, Nodal increased the mRNA levels of two steroidogenic enzymes hsd3b2 and cyp17a1, as well as paqr8, which encodes the membrane progestin receptor-β (mPR-β). Conversely, knockdown of ndr1 and ndr2 using siRNAs decreased the mRNA levels of hsd3b2, cyp17a1, and paqr8. Finally, treatment of Nodal significantly induced oocyte maturation. Taken together, these findings suggest that Nodal exerts multiple effects on zebrafish ovary to regulate follicle growth, steroidogenesis, and oocyte maturation.//////////////////The Interaction Between Nodal, Hypoxia-Inducible Factor 1 Alpha, and Thrombospondin 1 Promotes Luteolysis in Equine Corpus Luteum. Walewska E et al. (2019) The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.//////////////////
Nodal promotes functional luteolysis via downregulation of progesterone and prostaglandins E2 and promotion of PGF2 alpha synthetic pathways in mare corpus luteum. Galvão A et al. (2015) In the present work, we investigated the role of Nodal, an embryonic morphogen from TGFβ-superfamily, in corpus luteum (CL) secretory activity using cells isolated from equine CL as a model. Expression pattern of Nodal and its receptors ACVR2B, Alk7 and Alk4, as well as Nodal physiological role, demonstrate the involvement of this pathway in functional luteolysis. Nodal and its receptors were immune-localized in small and large luteal cells and endothelial cells, except ACVR2B, which was not detected in the endothelium. Nodal mRNA in situ hybridization confirmed its transcription in steroidogenic and endothelial cells. Expression analysis of the aforementioned factors evidenced that Nodal and Alk7 proteins peaked at the Mid-CL (p<0.01), the time of luteolysis initiation, while Alk4 and ACVR2B proteins increased from Mid- to Late CL (p<0.05). Nodal treatment of luteal cells decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p<0.05), as well as mRNA and protein of secretory enzymes StAR, CYP11A1, cPGES and mPGES1 (p<0.05). Conversely, PGF2α secretion and gene expression of PTGS2 and PGFS were increased after Nodal treatment (p<0.05). Mid-CL cells cultured with PGF2α had increased Nodal protein expression (p<0.05) and Smad3 phosphorylation (p<0.05). Finally, the supportive interaction between Nodal and PGF2α on luteolysis was shown to its greatest extent because both factors together more significantly inhibited P4 (p<0.05) and promoted PGF2α (p<0.05) synthesis than Nodal or PGF2α alone. Our results neatly pinpoint the sites of action of the Nodal signaling pathway towards functional luteolysis in the mare.//////////////////
ROLE AND REGULATION OF NODAL/ALK7 SIGNALING PATHWAY IN THE CONTROL OF OVARIAN FOLLICULAR ATRESIA. Wang H et al. Although the role of the transforming growth factor (TGF) beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by: (a) Co-localization of Nodal and its type I receptor ALK7 proteins in the granulosa cells was only observed in atretic antral follicles, while they were present in theca cells and granulosa cells of healthy follicles, respectively; (b) Addition of recombinant Nodal or over-expression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; (c) Constitutively active ALK7 (ALK7-ca) over-expression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, over-expression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased Xiap expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2 and cytochrome c in the granulosa cells. Dominant negative-Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, while up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.
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