ABSTRACT
Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.
Subject(s)
Activin Receptors, Type I/metabolism , Bone Morphogenetic Protein Receptors, Type I/metabolism , Mullerian Ducts/embryology , Mullerian Ducts/metabolism , Smad1 Protein/metabolism , Smad5 Protein/metabolism , Smad8 Protein/metabolism , Activin Receptors, Type I/deficiency , Activin Receptors, Type I/genetics , Animals , Anti-Mullerian Hormone/pharmacology , Bone Morphogenetic Protein Receptors, Type I/deficiency , Bone Morphogenetic Protein Receptors, Type I/genetics , Female , Infertility, Male/embryology , Infertility, Male/genetics , Infertility, Male/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Models, Biological , Mullerian Ducts/drug effects , Pregnancy , Signal Transduction , Smad1 Protein/genetics , Smad5 Protein/genetics , Smad8 Protein/geneticsABSTRACT
A region of homology, containing the contiguous SF3A2 (formerly called SAP62) and AMH genes, exists between human chromosome 19 (HSA19p) and mouse chromosome 10 (MMU10). In a previous study it was shown that SF3A2/Sf3a2 is very highly conserved between the two species and that AMH/Amh is somewhat less conserved although both human and mouse genes encode a protein (AMH) playing the same critical role during early male sex differentiation. The close association between SF3a2/Sf3a2 and AMH/Amh was thought to maintain open chromatin in the AMH/Amh promoter region, thus facilitating the necessary precise timing of AMH/Amh expression following that of SRY/Sry at the onset of testis differentiation. Further investigation of DNA upstream of Amh has revealed that there is another gene, in close association (about 400 bp) with Sf3a2, which has significant similarities to the N-terminus of a known guanine nucleotide releasing protein (GNRP) and consequently is provisionally named GNRPx/Gnrpx. The Gnrpx-Sf3a2-Amh (GSA) locus of the mouse (MMU10) is conserved in the human (HSA19p). Mapping the Sf3a2 transcription start site eventually led us to locate and characterize its promoter. We found that Sf3a2 and Gnrpx share a bi-directional promoter, with the latter being transcribed in an antisense direction. It has now been shown by RT-PCR analysis that both Sf3a2 and Gnrpx are widely expressed and therefore are likely to be 'housekeeping' genes. GNRPx/Gnrpx messenger RNA codes for a C-terminally truncated protein (149/164 aa), which contains an as yet uncharacterized domain common to GNRPs (and related proteins) and which may therefore act as a specific antagonist of a complete GNRP protein (>1200 aa) involved in the regulation of the GTPase (G-protein/Ras) cycle.
Subject(s)
Carrier Proteins/genetics , Glycoproteins , Growth Inhibitors/genetics , Guanine Nucleotide Exchange Factors/genetics , Nucleoproteins/genetics , Promoter Regions, Genetic/genetics , RNA-Binding Proteins , Testicular Hormones/genetics , Amino Acid Sequence , Animals , Anti-Mullerian Hormone , Base Sequence , COS Cells , DNA/chemistry , DNA/genetics , DNA, Antisense/genetics , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , RNA Splicing Factors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
Endothelial cells were isolated from bovine thoracic aorta and cultured. Bovine aortic endothelial cells (BAEC) were incubated with radiolabeled arachidonic acid (3H-AA) or eicosapentaenoic acid (14C-EPA) (1 microM) for 3 hr. Both fatty acids were predominantly incorporated into phosphatidylcholine (57 +/- 2% and 62 +/- 2% respectively) and slightly into phosphatidylethanolamine (11 +/- 0.5% and 12 +/- 0.6% respectively). phosphatidylinositol (26 +/- 1.5% and 10 +/- 0.5% respectively) and neutral lipids (6 +/- 0.5% and 15 +/- 1% respectively). After BAEC incubation with 3H-AA for 24 hr with or without EPA (1 microM), the release of radioactive metabolites of AA induced by thrombin (5.5 U/ml) was strongly reduced by the preliminary treatment with EPA (72 +/- 5%). After BAEC incubation with AA, EPA or vehicle (control), endothelin-1 levels were measured by RIA in the culture medium and we observed that: 1) the basal production of endothelin-1 was not modified after either AA or EPA treatment, 2) the thrombin-evoked release of endothelin-1 was significantly reduced by EPA (5.8 +/- 0.82 and 3.8 +/- 0.50 pg/microg proteins in control and EPA-treated cells, respectively); 3) by contrast, AA had no significant effect on the thrombin-evoked release of endothelin-1. In conclusion, EPA reduces strongly the endothelin-1 release but AA is ineffective. This reduction of endothelin-1 release may account partly for some of the vascular effects of EPA.
