Accidental X-Y recombination and the aetiology of XX males and true hermaphrodites

Accidental recombination between the differential segments of the X and Y chromosomes in man occasionally allows transfer of Y-linked sequences to the X chromosome leading to testis differentiation in so-called XX males. Loss of the same sequences by X-Y interchange allows female differentiation in a small proportion of individuals with XY gonadal dysgenesis. A candidate gene responsible for primary sex determination has recently been cloned from within this part of the Y chromosome by Page and his colleagues. The observation that a homologue of this gene is present on the short arm of the X chromosome and is subject to X-inactivation, raises the intriguing possibility that sex determination in man is a quantitative trait. Males have two active doses of the gonad determining gene, and females have one dose. This hypothesis has been tested in a series of XX males, XY females and XX true hermaphrodites by using a genomic probe, CMPXY1, obtained by probing a Y-specific DNA library with synthetic oligonucleotides based on the predicted amino-acid sequence of the sex-determining protein. The findings in most cases are consistent with the hypothesis of homologous gonad-determining genes, GDX and GDY, carried by the X and Y chromosomes respectively. It is postulated that in sporadic or familial XX true hermaphrodites one of the GDX loci escapes X-inactivation because of mutation or chromosomal rearrangement, resulting in mosaicism for testis and ovary-determining cell lines in somatic cells. Y-negative XX males belong to the same clinical spectrum as XX true hermaphrodites, and gonadal dysgenesis in some XY females may be due to sporadic or familial mutations of GDX.     

SRY-negative true hermaphrodites and an XX male in two generations of the same family

Two 46,XX true hermaphrodites and one XX male without genital ambiguities are reported. They coexist in two generations of the same pedigree, with paternal transmission and in the absence of SRY (sex-determining region, Y chromosome). These familial cases provide evidence to support the hypothesis that these disorders are alternative manifestations of the same genetic defect, probably an autosomal dominant mutation (with incomplete penetrance) or an X-linked mutation (limited by the presence of the Y chromosome).     

Females of four mole species of genus Talpa (Insectivora,mammalia) are true hermaphrodites with ovotestes

We studied the anatomical, histological, and genetic features of the sexual tract in four European mole species of the genus Talpa (Insectivora, mammalia): T. occidentalis, T. europaea, T. romana, and T. stankovici. All XY individuals had a normal male phenotype, whereas all XX individuals in all four species had features that identified them as intersexes. These individuals were nonetheless presumed to be functionally fertile females. Intersexuality was manifested mainly as gonadal hermaphroditism, with all females possessing bilateral ovotestes. The gonads were composed of a small portion of histologically normal ovarian tissue and a variably sized, generally large mass of disgenetic testicular tissue, accompanied by a small, rudimentary epididymis. The rest of the sexual tract was typically female, including oviducts, uterus, and vagina of normal appearance. Polymerase chain reaction (PCR) and Southern blotting analyses showed that the mammalian testis-determining gene SRY is present in males but not in females. Part of the conserved sequence of the mole SRY gene was cloned and sequenced after PCR amplification in two of the four mole species (T. occidentalis from Spain and T. romana from Italy). Sequences were identical in these two species and were very similar to those of the human and mouse SRY gene. Our findings constitute the first evidence of the existence of a genus-specific case of true hermaphroditism, probably due to a very ancient mutation that fixed in populations of the ancestral species from which contemporary moles evolved. The possible nature of this mutation is discussed with regard to the cytologic, histologic, and genetic features of the gonads in Talpa females. © 1996 Wiley-Liss, Inc.     

Absence of Y-specific DNA sequences in human 46,XX true hermaphrodites and in 45,X mixed gonadal dysgenesis

Summary A search for Y-specific DNA sequences has been performed in a sample of seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case and compared with a sample of 11 XX males. Using six Y-specific DNA probes no hybridization signal was obtained in the hermaphrodite group; in contrast, all XX males gave a positive signal with at least one probe. This difference is statistically highly significant. We conclude that the aetiology of true hermaphroditism is different from that of the XX male syndrome. As all cases of the hermaphrodite group are positive for the serological sex-specific antigen (Sxs) it is concluded that this antigen can be present even in the absence of Y-specific DNA.     

Genetic polymorphism of erythrocyte esterase-D in pigs

The erythrocyte esterase-D (Es-D) of four European-American pig breeds, two Taiwan native pigs (Taoyuan and Short ear breeds), Philippine native pig and wild boar populations (Sus scrofa riukiuanus) in Japan was investigated by using starch gel electrophoresis. Analysis of parentage records of the pigs revealed that the phenotypic variation of erythrocyte esterase-D was controlled by two codomi-nant alleles Es-D^A and Es-D^B. The allele Es-D^b was mostly found in three European -American pig breeds, Landrace (0.100), Hampshire (0.135) and Duroc (0.102). All of the wild boar populations were monomorphic for allele Es-D^A.     

Genetic relationship amongst the major non-coding regions of mitochondrial DNAs in wild boars and several breeds of domesticated pigs

We completed phylogenetic analysis of the major non-coding region of the mitochondrial DNA (mtDNA) from 159 animals of eight Euro-American and six East Asian domesticated pig breeds and 164 Japanese and five European wild boars. A total of 62 mtDNA haplotypes were detected. Alignment of these regions revealed nucleotide variations (including gaps) at 73 positions, including 58 sites with transition nucleotide substitutions, and two transversion substitutions. Phylogenetic analysis of the sequences could not organize domestic pig breeds into discrete clusters. In addition, many of the haplotypes found in members of diverged clustering groups were found primarily in Euro-American pig breeds, indicating extensive introgression of Asian domestic pigs into European breeds. Furthermore, phylogenetic analysis allocated the DNA sequences of non-coding regions into two different groups, and the deepest branchpoint of this porcine phylogeny corresponded to 86 000-136 000 years before present. This time of divergence would predate the historical period when the pig is thought to have been domesticated from the wild boar.     

Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes.

Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time-consuming procedures, and with them, we cannot "walk" chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end-specific "contextual" sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination with cloning of the inverse PCR-generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in "walking" chromosomes.     

Molecular characterization of the human ABO blood group orthologus system in pigs

The selection and use of animals with blood group 0 in the process of transplanting pig organs or tissues into humans can positively contribute to the control of acute immune rejection due to differences in blood groups. Exon-specific PCRs for the porcine blood group A transferase gene against genomic DNA from either blood group A or 0 animals resulted in the amplification failure of the A0 blood group gene exon 8 from blood group 0 animals. To characterize the genetic abnormality in the genome of blood group 0 animals, we screened bacterial artificial chromosome (BAC) clones from a Korean native pig BAC library which had the blood group 0 allele, and carried out shotgun sequencing. The analysis showed that the 0 allele has a large deletion between exon 7 of the A0 blood group gene and the neighbouring SURF6. We also showed that the ABO blood group antigens in humans and the A0 blood group antigens in pigs are coded by mutations within the orthologous glycosyltransferase gene. In addition, we developed a multiplex genotyping method for the porcine A0 blood group gene.     

Cytogenetic analysis of somatic and germinal cells from 38,XX/38,XY phenotypically normal boars

Many chromosomal abnormalities have been reported to date in pigs. Most of them have been balanced structural rearrangements, especially reciprocal translocations. A few cases of XY/XX chimerism have also been diagnosed within the national systematic chromosomal control program of young purebred boars carried out in France. Until now, this kind of chromosomal abnormality has been mainly reported in intersex individuals. We investigated 38,XY/38,XX boars presenting apparently normal phenotypes to evaluate the potential effects of this particular chromosomal constitution on their reproductive performance. To do this, we analyzed (1) the chromosomal constitution of cells from different organs in one boar; (2) the aneuploidy rates for chromosomes X, Y, and 13 in sperm nuclei sampled from seven XY/XX boars. 2n = 38,XX cells were identified in different nonhematopoietic tissues including testis (frequency, <8%). Similar aneuploidy rates were observed in the sperm nuclei of XY/XX and normal individuals (controls). Altogether, these results suggest that the presence of XX cells had no or only a very limited effect on the reproduction abilities of the analyzed boars.     

Genetic variability at the pig SLA complex in U.S. breeds of pigs1

Lymphocytes from 90 mature pigs and 548 growing pigs from 6.5 litters were screened with cytotoxic antibodies against swine lymphocyte antigens. Pigs included in the survey were from the Chester White, Duroc, Hampshire, Landrace and Yorkshire breeds. Significant differences between breeds were found in the frequency of the three haplotypes tested. Such differences may provide the genetic variability needed to determine associations between disease susceptibility and the pig's major histocompatibility gene complex.     

Skewed X-chromosome inactivation pattern in SRY positive XX maleness: a case report and review of literature

XX maleness is the most common condition in which testes develop in the absence of a cytogenetically detectable Y chromosome. Using fluorescence in situ hybridization (FISH) or PCR, it was possible to detect the transfer of Yp fragments including SRY gene to the terminal part of X chromosome in the majority of XX males. We report a 32-year-old-male in whom a seminal analysis showed azoospermia, an X chromatin analysis showed 44% of Barr body positive nuclei and a chromosomal analysis revealed a 46,XX karyotype. Physical examination showed a normal sexual development and bilateral small testes. Hormonal studies revealed hypergonadotropic hypogonadism. Testis histological examination showed a profile of Sertoli Only Cell Syndrome. FISH study ruled out the presence of a Y-bearing cell line, and confirmed translocation of SRY to Xp terminal part. In order to confirm that the complete masculinized phenotype was related to a preferential inactivation of the no rearranged X chromosome, X-chromosome inactivation patterns (XCIP) were studied by analysis of methylation status of the androgen receptor gene. Highly skewed XCIP was observed by greater than 90% preferential inactivation involving one of the two X chromosomes, suggesting that the SRY-bearing X chromosome was the preferentially active X allowing for sufficient SRY expression for complete masculinization.     

Divergence time estimates of East Asian and European pigs based on multiple near complete mitochondrial DNA sequences

The time since the divergence of European and East Asian domestic pigs and wild boars has been estimated in several phylogenetic analyses, generally based on partial mitochondrial sequences or on a small number of complete mtDNA sequences. In the present study, we obtained a refined estimate of this divergence time based on a set of 32 near‐complete mtDNA sequences from wild and domestic pigs of European and Asian types, including 14 new and 18 previously published sequences. A weighted average for different functional mtDNA components resulted in an estimate of 746 000 YBP for the divergence of Asian‐type from European‐type pigs. In addition, our data allowed us to estimate a divergence time between wild and domestic European pigs of 8500 YBP. However, it must be considered cautiously, as most of the estimated values of this sequence divergence were not different from zero, and isolation between wild and domestic pigs has never been complete.     

Genetic heterogeneity and selection signature at the KIT gene in pigs showing different coat colours and patterns

Mutations in the porcine KIT gene (Dominant white locus) have been shown to affect coat colours and colour distribution in pigs. We analysed this gene in several pig breeds and populations (Sicilian black, completely black or with white patches; Cinta Senese; grey local population; Large White; Duroc; Hampshire; Pietrain; wild boar; Meishan) with different coat colours and patterns, genotyping a few polymorphisms. The 21 exons and parts of the intronic regions were sequenced in these pigs and 69 polymorphisms were identified. The grey-roan coat colour observed in a local grey population was completely associated with a 4-bp deletion of intron 18 in a single copy KIT gene, providing evidence that this mutation characterizes the I^d allele described in the early genetic literature. The white patches observed in black Sicilian pigs were not completely associated with the presence of a duplicated KIT allele (I^p), suggesting that genetic heterogeneity is a possible cause of different coat colours in this breed. Selection signature was evident at the KIT gene in two different belted pig breeds, Hampshire and Cinta Senese. The same mutation(s) may cause the belted phenotype in these breeds that originated in the 18th–19th centuries from English pigs (Hampshire) and in Tuscany (Italy) in the 14th century (Cinta Senese). Phylogenetic relationships of 28 inferred KIT haplotypes indicated two clades: one of Asian origin that included Meishan and a few Sicilian black haplotypes and another of European origin.     

Quantitative analysis of squamous epithelium of normal palatal mucosa in guinea pigs

Summary The epithelium of intact guinea pig palate was subjected to stereologic analysis in a study of structural alterations in the keratinizing epithelium in response to wounding. Point counting procedures were employed to analyse electron micrographs sampled from three epithelial strata in biopsies collected from five animals. The differentiation pattern of the guinea pig palate epithelium displayed the following structural density gradients from basal to granular layers: descending gradients of metabolically active organelles, ascending gradient of bundled filaments coupled with the appearence of membrane coating granules and keratohyalin granules, and a plateau-like gradient of cytoplasmic ground substance. This pattern of epithelial differentiation is basically identical to that of human hard palate epithelium and epidermis. Regional and species variations in structure of keratinizing epithelia are suggested based on interepithelial differences in morphometric parameters.This investigation was supported in part by grant No. 512-4064 from the Danish State Medical Research Council and by a grant from the Calcin Foundation.The data recording and computation was performed on a guest visit at the Dental Institute, University of Zürich.     

小梅山、中梅山及大约克猪的SLA-DQB基因外显子2 PCR-RFLP多态性分析

采用限制性内切核酸酶Rsa I对中国地方猪种小梅山,中梅山及国外猪种大约克SLA－DQB基因外显子2的273bp扩增产物进行多态性分析,小梅山猪酶切后出现两种基因型：246bp/27bp(AA),132bp/84bp/30bp/27bp(BB),中梅山猪中出现4种基因型：246bp/27bp(AA),132bp/84bp/30bp/27bp(BB),132bp/114bp/27bp(CC),246bp/143bp84bp/30bp/27bp(AB),大约克猪中出现5 基因型;246bp/27bp(AA),142bp/84bp/30bp/27bp(BB),132bp/114bp/27bp(CC);246bp/132bp/84bp/30bp/27bp(AB).273bp/246bp/27bp(AD),分析发现,SLA－DQB基因外显子2的11,94和124位碱基表现出多态性并在3个猪种中检测到A,B,C和D4个等位基因,χ^2适应性检验结果表明,小梅山,中梅山及大约克SLA－DQB基因外显子2在Rsa I酶切位点 Hardy-Weinberg平衡状态。     

Sperm quality analysis in XX, XY and YY males of the Nile tilapia (Oreochromis niloticus)

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offspring), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology, such as growth, behavior and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm traits measured significantly differed between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males, respectively ranged from 0.92 to 1.33%, from 1.69 to 2.22 ×10⁹ cells mL⁻¹ and from 18′04″ to 27′32″. Mean values of total motility and curvilinear velocity 1 min after sperm activation, respectively ranged from 53 to 58% and from 71 to 76 μm s⁻¹ for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13% of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype.     

Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots

We have used bivariate flow karyotype analysis to quantify aberrant X chromosome size in 11 XX males. With one exception, the patients could be grouped into those with an X homologue difference greater than normal (Group A, n = 3) and into those whose X homologue difference could not be distinguished from female controls (Group B, n = 7). The range of sizes of the aberrant X chromosome in Y-sequence positive patients agrees with the variable nature of the X-Y interchange in these individuals as determined by the use of Y-specific DNA probes and Southern blotting analysis. In one patient it was possible to sort separately the normal and the X-Y interchanged homologues for dot blot analysis. The presence of Y sequences and an increased dose of the zinc finger gene, ZFY, were detected in the X-Y interchanged homologue. In preliminary studies of 5 male and 6 female controls, it was noted that a consistent difference between the two X homologues in females was found which could not be totally explained by errors of the fitting procedure. We suggest that this difference could be due to X inactivation and that the two X homologues in females might be distinguishable.