Josiane Szpirer

Alpha-fetoprotein protects the developing female mouse brain from masculinization and defeminization by estrogens

Two clearly opposing views exist on the function of alpha-fetoprotein (AFP), a fetal plasma protein that binds estrogens with high affinity, in the sexual differentiation of the rodent brain. AFP has been proposed to either prevent the entry of estrogens or to actively transport estrogens into the developing female brain. The availability of Afp mutant mice (Afp−/−) now finally allows us to resolve this longstanding controversy concerning the role of AFP in brain sexual differentiation, and thus to determine whether prenatal estrogens contribute to the development of the female brain. Here we show that the brain and behavior of female Afp−/− mice were masculinized and defeminized. However, when estrogen production was blocked by embryonic treatment with the aromatase inhibitor 1,4,6-androstatriene-3,17-dione, the feminine phenotype of these mice was rescued. These results clearly demonstrate that prenatal estrogens masculinize and defeminize the brain and that AFP protects the female brain from these effects of estrogens.

The gene map of the Norway rat (Rattus norvegicus) and comparative mapping with mouse and man

The current status of the rat gene map is presented. Mapping information is now available for a total of 214 loci and the number of mapped genes is increasing steadily. The corresponding number of loci quoted at HGM10 was 128. Genes have been assigned to 20 of the 22 chromosomes in the rat. Some aspects of comparative mapping with mouse and man are also discussed. It was found that there is a good correlation between the morphological homologies detectable in rat and mouse chromosomes, on the one hand, and homology at the gene level on the other. For 10 rat synteny groups all the genes so far mapped are syntenic also in the mouse. For the remaining rat synteny groups it appears that the majority of the genes will be syntenic on specific (homologous) mouse chromosomes, with only a few genes dispersed to other members of the mouse karyotype. Furthermore, the data indicate that mouse chromosome 1 genetically corresponds to two rat chromosomes, viz., 9 and 13, equalizing the difference in chromosome number between the two species. Further mappings will show whether the genetic homology will prove to be as extensive as these preliminary results indicate. As might be expected from evolutionary considerations, rat synteny groups are much more dispersed in the human genome. It is clear, however, that many groups of genes have remained syntenic during the period since man and rat shared a common ancestor. One further point was noted. In two cases groups of genes were syntenic in the mouse but dispersed to two chromosomes in rat and man, whereas in a third case a group of genes was syntenic in the rat but dispersed to two chromosomes in mouse and man. This finding argues in favor of the notion that the original gene groups were on separate ancestral chromosomes, which have fused in one rodent species but remained separate in the other and in man.

Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains.

In the HXB and BXH recombinant inbred strains derived from the spontaneously hypertensive rat and the normotensive Brown Norway rat, we determined the strain distribution patterns of 500 genetic markers to scan the rodent genome for quantitative trait loci regulating cardiac mass and blood pressure. The markers spanned approximately 1,139 cM of the genome and were tested for correlations with left ventricular mass adjusted for body weight, and with systolic, diastolic, and mean arterial pressures. The marker for the dopamine 1A receptor (Drd1a) on chromosome 17 showed the strongest correlation with left ventricular heart weight (P = .00038, r = -0.59) and the relationship to heart weight was independent of blood pressure. The markers showing the strongest correlations with systolic, diastolic, and mean arterial pressure were D19Mit7 on chromosome 19 (P = .0012, r = .55), D2N35 on chromosome 2 (P = .0008, r = .56), and Il6 on chromosome 4 (P = .0018, r = .53), respectively. These studies demonstrate that the HXB and BXH strains can be effectively used for genome scanning studies of complex traits and have revealed several chromosome regions that may be involved in the genetic control of blood pressure and cardiac mass in the rat.

A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility

The kidney has an important role in the regulation of acid–base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH3 diffusion coupled to H+ secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading—a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg-/- acid-loaded mice show reduced apical permeability to NH3 and impaired transepithelial NH3 transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

A genetic linkage map of the rat derived from recombinant inbred strains

We have constructed a genetic linkage map in the rat by analyzing the strain distribution patterns of 500 genetic markers in a large set of recombinant inbred strains derived from the spontaneously hypertensive rat and the Brown-Norway rat (HXB and BXH recombinant inbred strains). 454 of the markers could be assigned to specific chromosomes, and the amount of genome covered by the mapped markers was estimated to be 1151 centimorgans. By including a variety of morphologic, biochemical, immunogenetic, and molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH recombinant inbred strains.

Primary Structure, Tissue Distribution, and Chromosomal Localization of a Novel Isoform of Lysyl Hydroxylase (Lysyl Hydroxylase 3)

We report characterization of a novel isoform of lysyl hydroxylase (lysyl hydroxylase 3, LH3). The cDNA clones encode a polypeptide of 738 amino acids, including a signal peptide. The amino acid sequence has a high overall identity with LH1 and LH2, the isoforms characterized earlier. Conserved regions are present in the carboxyl-terminal portion of the isoforms and also in the central part of the molecules. Histidine and asparagine residues, which are conserved in the other isoforms and are known to be required for enzymatic activity, are also conserved in the novel isoform. The gene for LH3 (PLOD3) has been assigned to human chromosome 7q36 and rat chromosome 12. Gene expression of LH3 is highly regulated in adult human tissues. A strong hybridization signal, corresponding to an mRNA 2.75 kilobases in size, is obtained in heart, placenta and pancreas on multiple tissue RNA blots. Expression of the cDNAin vitro results in the synthesis of a protein that hydroxylates lysyl residues in collagenous sequences in a non-triple helical conformation.

Gene mapping in the rat by mouse-rat somatic cell hybridization: synteny of the albumin and alpha-fetoprotein genes and assignment to chromosome 14.

The methods of somatic cell genetics and molecular hybridization were applied to a panel of mouse X rat hepatocyte hybrids segregating rat chromosomes to assign the rat genes coding for two serum proteins, albumin and alpha-fetoprotein (Alb and Afp). The molecular hybridization of DNAs from different hybrids with cloned DNA probes showed that all the hybrid clones possessing the rat Alb gene and expressing it also retained the rat Afp locus, which is not expressed in these hybrids. So the Alb and Afp genes are syntenic in the rat, as in the mouse. Furthermore, the cytogenetic analysis allowed the assignment of these two loci to rat chromosome 14.

Mapping of the calcium-sensing receptor gene (CASR) to human chromosome 3q13.3-21 by fluorescence in situ hybridization, and localization to rat chromosome 11 and mouse chromosome 16.

The calcium-sensing receptor (CASR), a member of the G-protein coupled receptor family, is expressed in both parathyroid and kidney, and aids these organs in sensing extracellular calcium levels. Inactivating mutations in the CASR gene have been described in familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). Activating mutations in the CASR gene have been described in autosomal dominant hypoparathyroidism and familial hypocalcemia. The human CASR gene was mapped to Chromosome (Chr) 3q13.3-21 by fluorescence in situ hybridization (FISH). By somatic cell hybrid analysis, the gene was localized to human Chr 3 (hybridization to other chromosomes was not observed) and rat Chr 11. By interspecific backcross analysis, the Casr gene segregated with D16Mit4 on mouse Chr 16. These findings extend our knowledge of the synteny conservation of human Chr 3, rat Chr 11, and mouse Chr 16.

Genetic isolation of a region of chromosome 8 that exerts major effects on blood pressure and cardiac mass in the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of essential hypertension. Despite > 30 yr of research, the primary genetic lesions responsible for hypertension in the SHR remain undefined. In this report, we describe the construction and hemodynamic characterization of a congenic strain of SHR (SHR-Lx) that carries a defined segment of chromosome 8 from a normotensive strain of Brown-Norway rats (BN-Lx strain). Transfer of this segment of chromosome 8 from the BN-Lx strain onto the SHR background resulted in substantial reductions in systolic and diastolic blood pressure and cardiac mass. Linkage and comparative mapping studies indicate that the transferred chromosome segment contains a number of candidate genes for hypertension, including genes encoding a brain dopamine receptor and a renal epithelial potassium channel. These findings demonstrate that BP regulatory gene(s) exist within the differential chromosome segment trapped in the SHR-Lx congenic strain and that this region of chromosome 8 plays a major role in the hypertension of SHR vs. BN-Lx rats.

Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility

The alpha-fetoprotein gene (Afp) is a member of a multigenic family that comprises the related genes encoding albumin, alpha-albumin, and vitamin D binding protein. The biological role of this major embryonic serum protein is unknown although numerous speculations have been made. We have used gene targeting to show that AFP is not required for embryonic development. AFP null embryos develop normally, and individually transplanted homozygous embryos can develop in an AFP-deficient microenvironment. Whereas mutant homozygous adult males are viable and fertile, AFP null females are infertile. Our analyses of these mice indicate that the defect is caused by a dysfunction of the hypothalamic/pituitary system, leading to anovulation.