Contribution of domestic animals to the identification of new genes involved in sex determination.

Among farm animals, two species present an intersex condition at a relatively high frequency: pig and goat. Both are known to contain XX sex-reversed individuals which are genetically female but with a true hermaphrodite or male phenotype. It has been clearly demonstrated that the SRY gene is not involved in these phenotypes. Consequently, autosomal or X-linked mutations in the sex-determining pathway may explain these sex-reversed phenotypes. A mutation referred to as "polled" has been characterized in goats by the suppression of horn formation and abnormal sexual differentiation. The Polled Intersex Syndrome locus (PIS) was initially located in the distal region of goat chromosome 1. The homologous human region has been precisely identified as an HSA 3q23 DNA segment containing the Blepharophimosis Ptosis Epicanthus locus (BPES), a syndrome combining Premature Ovarian Failure (POF) and an excess of epidermis of the eyelids. In order to isolate genes involved in pig intersexuality, a similar genetic approach was attempted in pigs using genome scanning of resource families. Genetic analyses suggest that pig intersexuality is controlled multigenically. Parallel to this work, gonads of fetal intersex animals have been studied during development by light and electron microscopy. The development of testicular tissue and reduction of germ cell number by apoptosis, which simultaneously occurs as soon as 50 days post co?tum, also suggests that several separate genes could be involved in pig intersexuality.     

A reciprocal translocation, t(6p+; 14q-), in the pig

A reciprocal translocation, identified as t(6p+; 14q-), is described in a 38,XX intersex pig. It is the fourth reciprocal translocation to be reported for this species, whereas Robertsonian translocations, of frequent occurrence in cattle and sheep, are so far unknown in domestic pig breeds.     

The occurrence of C19 steroids in testicular tissue and submaxillary glands of intersex pigs in relation to morphological characteristics.

Five true hermaphrodite pigs and two male pseudohermaphrodite pigs were studied. A 38XX sex chromosome constitution was found in peripheral leucocytes of three true hermaphrodites and in one male pseudohermaphrodite; XX/XY mixoploidy was present in the leucocytes of the remaining male pseudohermaphrodite. The occurrence of C19 steroids, including 16-androstenes, in the testicular tissue and submaxillary gland of intersex pigs was of a similar pattern to that found previously in mature boars, and masculinization of the genital tract was related to the amount of testicular tissue present. It is postulated that in the absence of germ cells in the testicular tissue of intersex pigs the Sertoli cells may be involved in the metabolism of dehydroepiandrosterone to 5-androstenediol, a possible testosterone precursor in the pig. The high levels of 16-androstenes found in the submaxillary gland of intersex pigs indicates that these steroids are responsible for 'boar taint' in these animals. In contrast to the boar, no consistent relationship was found between the occurrence of C19 steroids and the degree of masculinization of the submaxillary gland; it is postulated that the predominantly female genetic constitution may have affected the response of the salivary gland to androgen.     

Molecular analysis in true hermaphroditism: demonstration of low-level hidden mosaicism for Y-derived sequences in 46,XX cases

True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.     

A case of an intersex horse with 63,X/64,XX/65,XX,del(Y)(q?) karyotype

Cytogenetic and molecular genetic studies of an intersex horse have been carried out. The investigated animal had overall male body conformation; however, its external genitalia consisted of incompletely developed vulva and penis. The X and Y chromosome painting probes detected three cell lines in the examined horse: 63,X, 64,XX and 65,XX with a fragment of a Y chromosome (del Y). The DNA analysis with the PCR and PCR/RFLP methods showed absence of SRY,AMELY and ZFY genes as well as of six Y microsatellite markers (YM2, YP9, YJ10, YE1, YH12, and YA16). These results suggest that the Y chromosome fragment detected in the investigated animal was the result of a deletion of a euchromatic fragment comprising the above-mentioned markers.     

Familial occurrence of pig intersexes (38,XX; SRY-negative) on a commercial fattening farm.

Occurrence of sex-reversal (38,XX; SRY-negative) cases in the progeny of a single boar was observed. Altogether 11 intersexes, originating from nine litters, given by nine sows were found. The breeder classified the sex-reversal individuals as females with enlarged clitoris. In addition, it was noticed that the anus was joined with the vulva. Moreover, in the scrotum-like structure one or two gonads were present. Cytogenetic evaluation was carried out for the sire, five dams and seven intersexes. The study revealed the normal male karyotype (38,XY) in the sire and the normal female karyotype (38,XX) in the dams and the intersexes. Molecular detection of the presence of the SRY gene was carried out for the sire, five dams, 10 intersexes and 28 phenotypically normal siblings. The SRY gene was present in the genotype of the sire and the male siblings. Three intersexes were subjected to detailed anatomical and histological examinations, after slaughter in a local slaughterhouse. Gonads were classified as testes with well-developed epididymis, however, without spermatogenetic activity. The presence of a properly developed uterus and ducti deferens was observed, but oviducts were not found. The collected data indicate that the sex-reversal status was caused by an unknown autosome, recessive mutation. Genetic background of this type of intersexuality is discussed in this study.     

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.     

SRY protein is expressed in ovotestis and streak gonads from human sex-reversal

In mammals, a master gene located on the Y chromosome, the testis-determining gene SRY, controls sex determination. SRY protein is expressed in the genital ridge before testis determination, and in the testis it is expressed in Sertoli and germ cells. Completely sex-reversed patients are classified as either 46,XX males or 46,XY females. SRY mutations have been described in only 15% of patients with 46,XY complete or partial gonadal dysgenesis. However, although incomplete or partial sex-reversal affects 46,XX true hermaphrodites, 46,XY gonadal dysgenesis, and 46,XX/46,XY mosaicism, only 15% of the 46,XX true hermaphrodites analyzed have the SRY gene. Here, we demonstrate that the SRY protein is expressed in the tubules of streak gonads and rete testis, indicating that the SRY protein is normally expressed early during testis determination. Based on these results, we propose that some factors downstream from SRY may be mutated in these 46,XY sex-reversal patients. We have also analyzed SRY protein expression in the ovotestis from 46,XX true hermaphrodites and 46,XX/46,XY mosaicism, demonstrating SRY protein expression in both testicular and ovarian portions in these patients. This suggests that the SRY protein does not inhibit ovary development. These results confirm that other factors are needed for complete testis development, in particular, those downstream of the SRY protein.     

Luteinizing hormone response to an oestradiol challenge in 5 intersex pigs possessing ovotestes

After challenge with oestradiol benzoate, the mean maximum LH concentration in 5 XX intersex pigs possessing ovarian and testicular tissue, or only testicular tissue, was 2.10 (+/- 0.41) ng/ml compared with 8.9 ng/ml in mature domestic gilts. These results indicate that exposure of the pig brain to testosterone before Day 30 of gestation is important, or that early testicular secretions other than testosterone are involved in the determination of brain gender. The observation that some intersex pigs show normal oestrous cycles implies that the response to these prenatal factors is primarily quantitative rather than qualitative.     

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.     

The human SRY protein is present in fetal and adult Sertoli cells and germ cells

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.     

Familial (9;11)(p22;p15.5)pat translocation and XX sex reversal in a phenotypic boy with cryptorchidism and delayed development

We describe a patient with the co-occurrence of a familial 9;11 reciprocal translocation and an XX sex reversal. The patient had cryptorchidism, delayed development, dysmorphic features and attention deficiency hyperactive disorder (ADHD). The proband's karyotype was 46,XX,t(9;11)(p22;p15.5) and he was positive for SRY gene. The father was found to be the carrier of the similar translocation. The co-occurrence of XX sex reversal and autosomal reciprocal translocation has not been described previously. The possible reasons for the manifestation of features other than those found in XX sex reversal is described.     

六例性反转综合征患者的分子遗传学分析

对六例性反转综合征患者(3例XX男性)(3例XY女性)用Y-特异性DNA探针进行了Southern印迹杂交分析,并用PCR技术扩增了SRY基因部分序列。结果表明,1例XX男性缺乏源于Y染色体的杂交信号,也无SRY基因;其余2例XX男性和3例XY女性都检测到Yp-DNA序列和SRY基因。这对进一步阐明性反转综合征的病因和SRY基因的作用机制具有重要意义。     

Complex mosaicism in sex reversed SRY+ male twins

Sex reversal is characterized by discordance between genetic and phenotypic sex. Most XX males result from an unequal interchange between X and Y chromosomes during paternal meiosis, therefore transferring SRY to the X chromosome, which explains the male development in the presence of an otherwise normal female karyotype. We present here the case of sex reversed SRY+ male twins with several cell lines. They consulted for infertility. The presence of SRY on an X chromosome was demonstrated by FISH. Their respective karyotypes were: 46,X,der(X)t(X;Y)(p22.3;p11.2)[249]/45,X [12]/45,der(X)t(X;Y)(p22.3;p11.2)[11]/47,XX,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1]/50, XX,der(X)t(X;Y)(p22.3;p11.2)x4[1]/46,XX[1] for the first twin (SH-1) and 46,X,der(X)t(X;Y)(p22.3;p11.2)[108]/45,X [3]/47,XX,der(X)t(X;Y)(p22.3;p11.2)[2]/45,der(X)t(X;Y) (p22.3;p11.2)[1]/47,X,der(X)t(X;Y)(p22.3;p11.2)x2[1] for the second twin (SH-2). There are three different types of XX males: 1) with normal genitalia, 2) with genital ambiguity, and 3) XX true hermaphrodites. The phenotype of the twins presented in this report is consistent with what is generally seen in XX SRY+ males: they have normal genitalia.     

Molecular cytogenetic analysis of XX males using Y-specific DNA sequences,including SRY

Summary XX maleness is the most common condition in which testes develop in the absence of a cytogenetically detectable Y chromosome. Using molecular techniques, it is possible to detect Yp sequences in the majority of XX males. In this study, we could detect Y-specific sequences, including the sex-determining region of the Y chromosome (SRY), using fluorescence in situ hybridization. In 5 out of 6 previously unpublished XX males, SRY was translocated onto the terminal part of an X chromosome. This is the first report in which translocation of an SRY-bearing fragment to an X chromosome in XX males could be directly demonstrated.     

SRY-negative XX sex reversal in a pony: a case report

A three year old pony with sexually ambiguous external genitalia was found to have a normal female karyotype (64, XX) and bilateral inguinal testes. The PCR analysis of blood samples revealed the absence of the Y chromosome sequences SRY, eTSPY and ZFY. No Y chromosome sequences were identified in DNA extracted from the gonads. The mechanism whereby XX sex reversal occurs in the absence of SRY is unknown.     

Expression of the human SRY protein during development in normal male gonadal and sex-reversed tissues.

Sex determination in mammals is controlled by the SRY gene located on the Y chromosome. It encodes a protein containing a DNA-binding and DNA-bending domain. In spite of recent advances in the identification of the mechanisms that regulate male sex determination in mammals, the expression profile of the SRY protein in normal and sex-reversed human tissues is not well established. In order to localize the SRY protein and determine its cellular distribution and expression at different stages of development, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One of these antibodies, LSRY1.1, recognizes a protein of 27 kDa in total lysates of HeLa SRYB3, a human cell line transfected with the SRY gene under the control of the SV40 promoter. Immunocytochemical analysis in the cell lines shows nuclear localization of the SRY protein. We have studied SRY protein expression in human tissues at different stage of fetal development until adult life and have demonstrated that the SRY protein is located in the nuclei of somatic cells and germ cells in the genital ridge during testis development. After testis determination, it can be detected until the adult stage in both germ cells and Sertoli cells. The presence of the SRY protein was also analyzed in biopsies of gonadal tissues of sex-reversal patients such as SRY-positive 46,XX males or SRY-positive 46,XX true hermaphrodites. SRY protein is detected in the nuclei of Sertoli cells of the testis and in the nuclei of granulosa cells in the ovotestis in these patients and in the nuclei of germ cells of both tissue types. These results suggest a common cellular origin for both Sertoli cells and granulosa cells.