XX/XY Chimerism in Internal Genitalia of a Virilized Heifer

SIMPLE SUMMARY: Freemartinism is the most common type of disorder of sex development (DSD) in heifers; it is caused by the formation of placental anastomoses between heterosexual twin fetuses and the transfer of masculine factors produced by the testes of the male co-twin to the female fetus. The abnormal development of external genitalia is commonly observed in such heifers, but it cannot be assumed that each heifer with ambiguous genitalia is an example of freemartinism. We genetically analyzed five DSD heifers, and four appeared to be freemartins, as revealed by the presence of XX/XY leukocyte chimerism. The fifth heifer had a normal XX sex chromosome complement and lacked the Y-chromosome-derived genes (SRY, ZFY and AMELY) in blood cells. This heifer was extensively studied through genetic, anatomical, and histological approaches. Postmortem anatomical and histological analysis showed the presence of normal ovaries, oviducts, and uterus, while three Y-linked genes (SRY, ZFY, and AMELY) were detected in DNA isolated from these organs. In conclusion, we suggest that among virilized heifers, there are, besides freemartins with XX/XY leukocyte chimerism, also cases with XX/XY chimerism in internal genitalia, the etiology of which remains unknown. ABSTRACT: Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY, AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.