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Comment |
[P2-139] Identification of the Anti-Mullerian Hormone Signaling Pathway in the Ovary.
Jenny A Visser, Maria JG Gruijters, Anke McLuskey, Mark Cranfield, Nigel P Groome, Axel PN Themmen Dept of Intern Med, Erasmus MC, Rotterdam, Netherlands; Sch of Pharmacol and Molec Scis, Oxford Brookes Univ, Headington, Oxford, United Kingdom
Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a member of the transforming growth factor (TGF) superfamily and plays a crucial role during male sexual differentiation. In addition, AMH has an important function in the ovary where it inhibits the recruitment of primordial follicles into the growing follicle pool and decreases the sensitivity of growing follicles to FSH. However, the molecular mechanisms by which AMH elicits its effects on the ovary are unknown.
TGF family members signal through a heteromeric receptor complex of type II and type I serine/threonine kinase receptors, which activate downstream Smad proteins. For AMH one specific type II receptor has been identified. We have shown that, in the Mullerian duct, AMH induces a bone-morphogenetic protein (BMP)-like response mediated through the BMP type I receptor ALK2. Others have shown that, besides ALK2, the other BMP type I receptors ALK3 and ALK6 are also involved in AMH signaling.
To address the AMH signaling pathway in the ovary we use KK-1 cells, a mouse granulosa cell line. AMH induces the BRE3-Luc reporter, a luciferase reporter containing three BMP response elements, after transient transfection of AMHRII. In contrast, AMH did not induce the TGF-responsive 3TP-lux reporter, suggesting that also in KK-1 cells AMH induces a BMP-like signaling pathway. In agreement with this, we found that AMH induces the phosphorylation of Smad1. These results suggest that KK-1 cells are an excellent tool to study the molecular mechanisms by which AMH mediates its effect on the ovary.
In addition, we have generated a KK-1 cell-line stably expressing the AMHRII. In these cells we have tested the neutralizing effect of newly developed AMH monoclonal antibodies on AMH-induced activation of the BRE3-luc reporter. Of the six antibodies tested, two were able to inhibit AMH signaling in a dose dependent manner. Furthermore, we have used the KK-1/AMHRII cell line to identify AMH-regulated genes. Many TGF family members upregulate the expression of the inhibitory Smad 6 and 7, thereby generating a negative feedback loop. In agreement, we found that, in the KK-1/AMHRII cells, AMH strongly induces the expression of Smad6 (10-fold) and to a lesser extent that of Smad7 (2.5-fold). Thus, Smad6 and 7 are one of the first AMH-regulated genes in the ovary.
Experiments are ongoing to identify additional AMH-regulated genes using microarray technology.
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Mutations |
1 mutations
Species: ovine
Mutation name:
type: naturally occurring
fertility: fertile
Comment: Trio, a novel high fecundity allele: I. Transcriptome analysis of granulosa cells from carriers and non-carriers of a major gene for bovine ovulation rate. Kamalludin MH et al. (2017) A major gene for bovine ovulation rate has been mapped to a 1.2 Mb region of chromosome 10. Screening of coding regions of positional candidate genes within this region failed to reveal a causative polymorphism, leading to the hypothesis that the phenotype results from differences in candidate gene expression rather than alteration of gene structure. This study tested differences in expression of positional candidate genes in granulosa cells between carriers and non-carriers of the high fecundity allele, as well as characterizing differences in the transcriptomic profile between genotypes. Five carriers and five non-carriers, female descendants of "Trio," a carrier of the high fecundity allele were initially used in an RNA-seq analysis of gene expression. Four of ten samples were contaminated with theca cells, so that six samples were used in the final analysis (three of each genotype). Of 14,973 genes expressed, 143 were differentially expressed (false discovery rate p < 0.05) in carriers versus non-carriers. Among the positional candidate genes, SMAD6 was 6.6 fold over expressed in the carriers compared to non-carriers (p < 5 × 10-5). This result was replicated in an independent group of 12 females (7 carriers, 5 non-carriers) using quantitative real-time PCR; SMAD6 was 9.3 fold overexpressed in carriers versus non-carriers (p = 1.17 × 10-6). Association of overexpression of SMAD6, an inhibitor of the BMP/SMAD signaling pathway, with high ovulation rate corresponds well with disabling mutations in ligands (BMP15, GDF9) and a receptor (BMPR1B) of this pathway that cause increased ovulation rate in sheep.//////////////////
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