XX/XY Chimerism in Internal Genitalia of a Virilized Heifer

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.

Keywords: AMELX; AMELY; SOX9; SRY; XX/XY chimerism; ZFX; ZFY; cattle; disorder of sex development; freemartinism; intersexuality.

Figure 1

Virilized external genitalia of studied cases. (a) Case #7497. (b) Case #7502. (c) Case #7515. (d) Case #7518. (e,f) Case #7514.

Figure 2

Representative metaphase spreads derived from in vitro leukocyte culture of DSD heifer #7514: (a) Giemsa staining—submetacentric X chromosomes are indicated by arrows, (b) C-banding—X chromosomes with no centromeric positive C band block are indicated by arrows, (c) G-banding—X chromosomes with normal patterns are indicated by arrows and enlarged in a right down corner. Scale bar = 10 µm.

Figure 3

Anatomical and histological analysis of the DSD heifer #7514: (a) internal genitalia—uterus with oviducts and ovaries; (b) Graafian follicle with an oocyte surrounded by granulosa cells, scale bar = 200 µm; (c) cross section of the oviduct: folded mucosa and thin muscularis, scale bar = 500 µm; (d) uterine mucosa with small endometrial glands in the basal layer and tubular glands in the functional layer, scale bar = 500 µm. Corpus luteum (CL) and ovarian follicle (OF) are indicated by arrows.

Figure 4

Detection of SRY (a) and ZFY (b) genes in DSD heifer #7514. L: GeneRuler DNA ladder; B: blood; H: hair follicles; O1: ovary 1; O2: ovary 2; U: uterus; OD: oviduct; M: control male; FM: control female; NC: negative control (no DNA).

Figure 5

Genotypes for selected microsatellite markers in different tissues of DSD heifer #7514: TGLA227 (a) and TGLA53 (b). Three size variants for TGLA227 (83, 89 and 103 bp) and TGLA53 (160, 162 and 170) in internal genitalia, instead of two variants observed in blood cells and hair follicles, are visible.

Figure 6

Estimation of the Y/X ratio by ddPCR based on the proportion of AMELY (Ch2) and AMELX (Ch1) amplicons in the DSD heifer #7514. (a) Amplification signals from chromosome X (blue color) and Y (green color). (b) Y/X ratio is presented. B: blood; H: hair follicles; O1: ovary 1; O2: ovary 2; U: uterus; OD: oviduct; M: control male; FM: control female; NC: negative control (no DNA).