A 11.7-kb deletion triggers intersexuality and polledness in goats.

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Cloning and characterization of a full-length cDNA coding for ovine aldolase B from fetal mesonephros.

An ovine aldolase B cDNA was isolated from mesonephros (29 d pc). The sequence covers 1649 nucleotides. Comparison with human liver aldolase B cDNA shows a homology of about 86%. The deduced amino acid sequence is composed of 364 residues and exhibits 92% homology to the human protein. Northern blot analysis and in situ hybridization data show that during the first third of gestation in sheep, aldolase B expression is restricted to the mesonephros.

Cloning of a teleost fish glucocorticoid receptor shows that it contains a deoxyribonucleic acid-binding domain different from that of mammals.

In the teleost fish, physiological and biochemical studies suggest that glucocorticoids regulate both salt balance and metabolic activities. In mammals, however, these functions are divided between glucocorticoids and mineralocorticoids. In mammals, separate receptors for these two classes of steroid hormone have been cloned and sequenced. To begin to understand the regulation in fish of the vital processes ascribed to glucocorticoids, we have cloned, sequenced, expressed, and studied the steroid-binding and transcriptional activation capabilities of the rainbow trout (Onchorhynchus mykiss) glucocorticoid receptor. Northern blot analysis shows a single rainbow trout GR messenger RNA species of 7.5 kilobases expressed in gill, intestine, skeletal muscle, kidney, and liver. The trout GR 2274-nucleotide coding sequence provides for a protein of 758 amino acids, with appropriate similarities to mammalian GR, with one striking exception. As in other members of the steroid/thyroid/retinoid receptor family, the DNA-binding domain contains two putative zinc fingers. These have high homology with those of other GRs. However, between the zinc fingers in the trout GR are found 9 more amino acids than are seen in mammalian GRs, raising questions as to the functional form of the fish, as opposed to the mammalian, GR. It has been proposed that as fish appear to use glucocorticoids for both metabolic and salt control, presumably through a single GR, GR would prove to be the evolutionary precursor to mammalian GR and mineralocorticoid receptor (MR). Computer analysis of the known sequences of GRs and MRs, however, suggests that the fish GR did not give rise to the MR of higher animals, but that both subfamilies of receptor arose from some earlier gene.

Expression studies of the PIS-regulated genes suggest different mechanisms of sex determination within mammals.

In mammals, the Y-located SRY gene is known to induce testis formation from the indifferent gonad. A related gene, SOX9, also plays a critical role in testis differentiation in mammals, in birds and reptiles. It is now assumed that SRY acts upstream of SOX9 in the sex determination cascade, but the regulatory link which should exist between these two genes remains unknown. Studies on XX sex reversal in polled goats (PIS mutation: Polled Intersex Syndrome) have led to the discovery of a female-specific locus crucial for ovarian differentiation. This genomic region is composed of at least two genes, FOXL2 and PISRT1, which share a common transcriptional regulatory region, PIS. In this review, we present the expression pattern of these PIS-regulated genes in mice. The FOXL2 expression profile of mice is similar to that described in goats in accordance with a conserved role of this ovarian differentiating gene in mammals. On the contrary, the PISRT1 expression profile is different between mice and goats, suggesting different mechanisms of the primary switch in the testis determination process within mammals. A model based on two different modes of SOX9 regulation in mice and other mammals is proposed in order to integrate our results into the current scheme of gonad differentiation.

Lipoplex-induced hemagglutination: potential involvement in intravenous gene delivery.

We report a study aiming to characterize the interaction of blood and blood components with lipoplexes under conditions relevant to in vivo intravenous transfection. In this study we focus on the interaction of lipoplexes with red blood cells (RBC). It was found that no significant hemolysis occurred during several hours' incubation using lipoplex compositions and lipoplex/red blood cell ratios in the range commonly used for in vivo transfection. However, the interaction of RBC with lipoplexes resulted in massive agglutination, which occurs irrespective of the type of cationic lipid or helper lipid. Agglutination was also induced by polyplexes (such as dendrimer/DNA complexes) and lipoplexes in the presence of spermidine or protamine sulfate (the latter induced hemagglutination by itself). DSPE-PEG(2000) inserted into the lipoplexes inhibits hemagglutination somewhat. In order to understand the effect of serum on the agglutination better, plasma was separated into its high molecular weight components (HMWC, >14 kDa) and its low molecular weight components (LMWC, < or = 14 kDa). These fractions were characterized for their level of proteins, primary amino groups, osmotic pressure, and electrical conductivity, and compared with saline (0.15 M NaCl). It was found that both LMWC and HMWC inhibit agglutination by themselves, although whole serum demonstrates better hemagglutination inhibition than each fraction separately. The inhibitory effect of the serum (or plasma) is explained by its effect on the electrostatics of the lipoplexes, reducing their positive charge, as was demonstrated using fluorescein-phosphatidylethanolamine-labeled lipoplexes. The effect of LMWC was related to ionic strength and was equal to the effect of 0.15 M NaCl. The level of agglutination was reduced with increasing lipoplex DNA(-)/cationic lipid(+) (DNA(-)/L(+)) ratio. However, at the low DNA(-)/L(+) ratio needed to achieve significant in vivo transfection after i.v. administration, massive agglutination occurred. These data suggest that i.v. administration of lipoplexes and polyplexes may lead to RBC agglutination, and the agglutinates formed may explain the localization of lipoplexes and expression of their transgenes in the lungs.

Time course of female-to-male sex reversal in 38,XX fetal and postnatal pigs.

In an attempt to understand the etiology of intersexuality in pigs, we thoroughly analyzed the gonads of 38,XX (SRY negative) female to male sex-reversed animals at different developmental stages: during fetal life [50 and 70 days postcoitum (dpc)], just after birth [35 days postpartum (dpp)] and during adulthood. For each animal studied, we performed parallel histological and ultrastructural analyses on one gonad and RT-PCR analysis on the other gonad in order to define the expression profiles of sexually regulated genes: SOX9, 3beta-HSD, P450 aromatase, AMH, FOXL2, and Wnt4. Light and electron microscopic examination showed that testicular cords differentiated in XX sex-reversed gonads but were hypoplastic. Although the testicular cords contained gonia at the fetal stages, the germ cells had all died through apoptosis within a few weeks after birth. Ultrastructurally normal Leydig cells also differentiated, but later, and enclosed whorl-like residual bodies. At the fetal stages, three of the six genes studied in the intersex gonads presented, as early as 50 dpc, a modified expression profile corresponding to an elevated expression of SOX9 and the beginning of AMH and P450 aromatase gene transcription. In addition to genes involved in the testicular pathway, the same gonads expressed FOXL2, an ovarian-specific factor. The ovaries of true hermaphrodites were ineffective in ensuring correct folliculogenesis and presented abnormal expression profiles of ovarian specific genes after birth. These results indicate that the genes involved in this pathology act very early during gonadogenesis and affect the ovary-differentiating pathway with variable expressivity from ovarian germ cell depletion through to trans-differentiation into testicular structures.