Pietro Parma

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, 3β‐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.

Relevance of intersexuality to breeding and reproductive biotechnology programs; XX sex reversal in pigs

Abstract In pigs, the frequency of intersexuality ranges from 0.1 to 0.6%. Economic losses are significant and result from sterility, urogenital infections and downgrading of carcasses because of boar taint. To elucidate the genetic aetiology of pig sex reversion, 50 intersexes from a closed breeding herd were analysed. The majority have a 38,XX karyotype without Y chromosome sequences. Phenotypically, the animals range from true hermaphrodites to XX males with or without genital ambiguities. Analysis of crosses between parents of intersexes showed that the trait is inherited and caused by the same mutation in the Large White breed. Investigations are currently being carried out to isolate a genetic marker linked to intersexuality using genetic map of the porcine genome. Such a marker will be useful in eradicating the mutation in pig breeding herds and in permiting the isolation of the gene responsible.

Porcine Dax-1 gene: isolation and expression during gonadal development.

The identification of XY females carrying a duplication of a region of the X chromosome (Xp21) led to the hypothesis that a double dose of a gene in the duplicated region causes sex reversal (DSS; dosage sensitive sex reversal). A gene isolated from this region, named DAX-1 (DSS-AHC critical region on the X), encodes a new member of the nuclear hormone receptor family. Here, we describe the isolation of porcine Dax-1 and the analysis of its pattern of expression both during foetal development and in several adult tissues. Dax-1 is expressed in the adrenals, the pituitary gland and the gonads at various stages of differentiation. In gonads, Dax-1 expression starts between 21 and 23 days post coitum in both XX and XY urogenital ridges then continues to be expressed until adult age. The expression in these tissues indicates the involvement of DAX-1 in the development and the function of the reproductive system at multiple levels.

Sex Reversal in Non-Human Placental Mammals

Gonads are very peculiar organs given their bipotential competence. Indeed, early differentiating genital ridges evolve into either of 2 very distinct organs: the testis or the ovary. Accumulating evidence now demonstrates that both genetic pathways must repress the other in order for the organs to differentiate properly, meaning that if this repression is disrupted or attenuated, the other pathway may completely or partially be expressed, leading to disorders of sex development. Among these disorders are the cases of XY male-to-female and XX female-to-male sex reversals as well as true hermaphrodites, in which there is a discrepancy between the chromosomal and gonadal sex. Here, we review known cases of XY and XX sex reversals described in mammals, focusing mostly on domestic animals where sex reversal pathologies occur and on wild species in which deviations from the usual XX/XY system have been documented.

Testicular XX (SRY-Negative) Disorder of Sex Development in Cat

In most mammals, the sex of an individual is genetically determined by the Y chromosome-specific SRY gene. The presence of at least one functional copy of this gene determines the development of the primordial gonads into testes. However, testicular tissue does develop in the absence of SRY, albeit rarely, which is the case in testicular XX (SRY-negative) disorder of sex development (DSD). This condition is very important for studying the process of sexual determination because it allows the identification of genetic factors that are able to promote the male developmental pathway in the absence of SRY and thereby enables a better understanding of this process. Until now, this condition has been identified in various animal species but has never been reported in cat. In this study, we describe the first case of an XX (SRY-negative) DSD cat. The cat possesses a tortoiseshell coat associated with male-like external genitalia, including normal scrotum with 2 palpably normal testicles. Histological analysis confirmed the presence of the testes, and cytogenetic and genetic analyses showed a female karyotype associated with the absence of the SRY gene. Finally, sequencing of the RSPO1 gene revealed no mutation, and FISH analysis of the SOX9 locus did not reveal any large abnormalities.

Centromere Repositioning in Cattle (Bos taurus) Chromosome 17

Eukaryotic organisms have developed a structure, called centromere, able to preserve the integrity of the genome during cell division. A young bull from the Marchigiana breed, with a normal external phenotype, underwent routine cytogenetic analysis to enter the reproduction center. All metaphases analyzed showed an unusual biarmed chromosome of medium size despite a diploid set of chromosomes (2n = 60,XY). FISH analysis excluded a pericentric inversion or a reciprocal translocation, but highlighted a repositioning of the centromere in BTA17. The satellite DNA was still in an acrocentric position. The telomeres were normally present. The primary constriction on the abnormal chromosome was C-band negative. Finally, the absence of a large genomic deletion in the BTA17 pericentromeric region was demonstrated by both array-CGH analysis and SNP array. To our knowledge, this is the first case of centromere repositioning reported in cattle.

XY ( SRY -positive) Ovarian Disorder of Sex Development in Cattle

In mammals, the sex of the embryo depends on the SRY gene. In the presence of at least one intact and functional copy of this genetic factor (XY embryo) undifferentiated gonads will develop as testicles that subsequently determine the male phenotype. When this factor is not present, i.e., in subjects with 2 X chromosomes, an alternative pathway induces the development of ovaries, hence a female phenotype. In this case study, we describe a female cattle affected by a disorder of sex development (DSD). The subject, despite having a chromosomal XY constitution, did not develop testicles but ovaries, although they were underdeveloped. Moreover, genetic analysis highlighted the presence of the SRY gene with a normal coding region in both blood- and tissue-derived DNA. A chimeric condition was excluded in blood by sexing more than 350 cells and by allele profile investigation of 18 microsatellite markers. Array CGH analysis showed the presence of a not yet described 99-kb duplication (BTA18), but its relationship with the phenotype remains to be demonstrated. Gonadal histology demonstrated paired ovaries: the left one containing a large corpus luteum and the right one showing an underdeveloped aspect and very few early follicles. To our knowledge, we describe the first case of XY (SRY+) DSD in cattle with a normal SRY gene coding sequence.

Persistent Müllerian Duct Syndrome in a German Shepherd Dog

In mammals, the regression of the müllerian ducts is regulated by the action of the AMH hormone which is produced by testes during embryonic development. The action of this hormone is mediated by the only known receptor AMHR2. Mutations occurring in the AHM hormone and/or in the AMHR2 receptor gene cause the lack of regression of müllerian ducts, which may therefore persist even in male embryos carrying a XY chromosomal arrangement. This is known as the persistent müllerian duct syndrome (PMDS). A female German Shepherd dog was referred to the veterinary clinic because of urinary incontinence. She also showed an anatomical structure that protruded from and enlarged the vulvar labia. From the morphological appearance, one gonad resembled an ovary and the other a testicle. The histological examination instead showed that the gonads were both testes with an underdeveloped parenchyma and without signs of spermatogenetic activity. No alterations were found with regard to the uterus which showed a correctly developed body, cervix, and horns. Genetic analysis, performed on DNA extracted from blood, showed (i) the presence of both X and Y chromosomes, (ii) the absence of chromosome XX/XY chimerism, (iii) a normal SRY gene coding sequence, (iv) a normal AMHR2 gene coding sequence, and (v) a normal AMH gene coding sequence. In this study, we report and characterize a new case of PMDS in a dog excluding that the only mutation hitherto found in the AMHR2 gene is responsible for the observed phenotype.

Contents Vol. 151, 2017

Plant cytogenetics and genomics Andreas Houben Institute of Plant Genetics and Crop Plant, Research (IPK) Corrents-Str. 3 Gatersleben, D–06466 (Germany) Tel. (+1) 785 532 2364; Fax (+1) 785 532 5692 E-mail: houben@ipk-gatersleben.de Tumor cell genetics and cancer cytogenetics Ad Geurts Van Kessel Department of Human Genetics University Hospital P.O. Box 9101 NL–6500 HB Nijmegen (The Netherlands) Tel. (+31) 24 361 4107; Fax (+31) 24 354 0488 E-mail: a.geurtsvankessel@antrg.umcn.nl

Contents Vol. 10, 2016

Karin Schmid (address as for M. Schmid) E-mail: karin.schmid@uni-wuerzburg.de Peter Koopman Professor of Developmental Biology Institute for Molecular Bioscience The University of Queensland AU–Brisbane, Qld. 4072 (Australia) Tel. (+61) 7 3346 2059; Fax. (+61) 7 3346 2101 E-mail p.koopman@imb.uq.edu.au Manfred Schartl Institute of Physiological Chemistry I University of Würzburg Biozentrum, Am Hubland D–97074 Würzburg (Germany) Tel. (+49) 931 318 4148; Fax (+49) 931 318 4150 E-mail: phch1@biozentrum.uni-wuerzburg.de