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Introduction

     Haemopoietic chimerism has received special interest from researchers in bovine cytogenetics due to the freemartin condition which is characterized by virilization signals in a female calf born twin with a male. A large number of XX cells at mitosis in the testes of newborn calves, twins of females, was found (Ohno et al., 1962). It was demonstrated that germ cells might also be exchanged by placental anasthomosis fusion and Herschler and Fechheimer (1967) proposed that the freemartin condition was due to the presence of XY cells in the female blood.

    By in situ hybridization with fluorescence (FISH) for chromosome identification, Rejduch et al. (2000) studied three mature bulls born in heterosexual twinning and demonstrated spermatogonial chimerism, indicating the possibility of survival and differentiation of XX cells in the germ cell lines.

    Marmosets usually have twins or triplets enclosed in a single chorion resulting in a natural haemopoietic chimerism (Hill and Hill, 1927). Despite the similarity of this placentation with that of bovine twins, the freemartin effect was not produced (Wislocki, 1939).
The anastomoses between marmoset twins lead to permanent blood chimerism, which was demonstated using sex chromosome differences in leucocyte cultures from blood (Benirschke et al, 1962). However, natural chimerism without the freemartin condition seen in other species is still an enigma because it allows hematopoietic and bone marrow chimerism even if it does not result in freemartinism between twins of different sexes. Thus primates differ from cattle in which the freemartin animal is frequently infertile and virilized (Wislocki, 1939).  Sex determination in mammals is genetically controlled, while sex differentiation is controlled by endocrine events. Male sex determination in mammals has been associated with the highly conserved sex determining region Y (SRY) gene (Sinclair et al., 1990).
Studies in marmosets (Hearn, 1982) indicate that during late pregnancy, male and female fetuses have similar levels of blood testosterone although the female is apparently unaffected in its gonadal or behavioural development.

    The aim of this work is to study a female Leontopithecus chrysomelas with XX/XY chimerism and virilization signals, and discuss this novel condition.

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Materials and Methods

    A ten year old female Leontopithecus chrysomelas, reared in captivity in the Primatology Center of Rio de Janeiro (CPRJ-FEEMA) located in the city of Guapimirim, Rio de Janeiro State, Brazil was studied. The animal showed an enlarged clitoris, constriction of the vulvar opening and absence of reproductive behaviour.

    The cytogenetic investigation was carried out in metaphases obtained from lymphocyte cultures (Moorhead et al, 1960), stained by conventional staining of chromosomes with giemsa 3% and G- banding (9).
Surgical gonadectomy and a biopsy were performed. Some of the material was processed for in vitro culture to get metaphases and the other part for histological study.

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Results

    Clinical examination of the animal revealed a siginificant clitoris enlargement (fig. 1) and constriction of the vulvar opening. The chromosome complement found was represented by two cell lineages with 80% 46,XX(fig.2) and 20% 46,XY (fig.3). During the surgical procedure the presence of ovaries, uterus and uterine horns with a normal female appearance was observed. Histological study of the two gonads confirmed ovarian pattern tissue characterized by oocytes enveloped by antral follicles, and the presence of corpora lutea in the right ovary indicating ovulation.
The tissue culture technique for obtaining metaphases from the ovaries did not provide satisfactory material.
 

Figure 1 - Female Leontopithecus chrysomelas presenting enlarged clitoris.

Figure 2 - Karyotype of metaphase with 46,XX chromosomes from a female Leontopithecus chrysomelas.

Figure 3 - Karyotype of metaphase with 46,XY chromosomes from a female Leontopithecus chrysomelas.

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Discussion and conclusions

    Chromosome chimerism between species from the Callitrichidae family has been frequently described as a normal situation in twinning pregnancies, unlike what occurs in cattle which present alterations of sex differentiation due to XY cells in the undifferentiated female gonad. Benirschke et al. (1962) studied femoral bone marrow from three adult marmosets, two male Callithrix jacchus and one female Leontocebus rosalia, and found metaphases from the opposite sex.

    Benirschke and Brownhill (1963) described the presence of XX spermatogonias in the male testes from three different species of marmoset. Sanchez-Morgado et al (2003) reported a marmoset (Callithrix jacchus) of atypical external phenotypic genitalia appearence, twin to a male and a female and having a single opening for the urethra in the genitalia without evidence of a normal vulva or testes. At necropsy, normal uterus and ovaries were found with no evidence of residual testicular tissue. It was documented to carry Y chromosome sequences ZFY and SRY. The SRY gene exon did not contain mutations or deletions, indicating that the presence of a normal SRY exon does not prevent the development of ovaries, uterus, and vagina in marmosets. Had it been a chimeric female, Y sequences should be present. Ryan at al (1961) suggested, for the first time, that the primate placenta presents an enzymatic constitution able to convert the male gonadal hormones into estrogens.

    The complete sequence of cDNA from enzyme 17{beta}-hydroxysteroid dehydrogenase from marmosets including the proximal promoter region and the partial sequence of aromatase from marmosets were sequenced with the objective of identifying tissues which participate in the synthesis of estradiol steps, demonstrating similarity with human. Northern blots showed preferential expression in the placenta. An enzymatic conversion from testosterone to estradiol was found in the corpora lutea of marmosets. The coexpression pattern with aromatase, supports the hypothesis that 17HSD7 ensures the continuation of the estradiol synthesis throughout gestation in marmosets (Husen et al, 2003).

    The animal studied here presented virilization of external genitalia, characterized by enlarged clitoris and constriction of vulvar opening. Macroscopically and microscopically, all tissues analysed, including the gonads, were normal for this species and were those of the female sex. Enlargement of the clitoris is a consequence of testosterone production although no testicular tissue was found.
XX males range phenotypically from completely virilized individuals to true hermaphrodites and include a subset of SRY negative subjects. The correlation between genotype (SRY+/-) and phenotype is still unclear (Grigorescu-Sido et al., 2005).
In cattle and other mammals, the freemartin condition is due to the presence of XY cell population which settles in the female gonad with the expression of Sry gene, responsible for testis determination at an early phase of gonadal differentiation. The chimerism between marmoset twins of different sexes was demonstrated only in the bone marrow and male gonads. Although the reverse condition has not been reported in primates, it suggests the necessity of investigating the presence of XY cells in the female gonad and the Sry expression in this tissue.

    Esser et al. (1981), studied true hermaphroditism in a child with XX/XY cell lineage. They could not find tissue of the testis but confirmed the diagnosis by detection of the H-Y antigen and by typical endocrinological findings.
In this case, exogenous testosterone production during gestation is very improbable and an adrenal hyperplasia which could have presented specific symptoms was absent.

    Thus, the present case suggests that despite testicular tissue was undetectable in the gonads, a minimal amount would be enough to produce testosterone resulting in an enlarged clitoris, as evidence for a true hermaphroditic condition.
 

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References

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