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.


Introduction
Freemartinism is the most common type of disorder of sex development (DSD) in cattle. It is caused by a transfer of masculine factors from a male fetus to a co-twin female fetus through placental anastomoses. This abnormality is classified as a sex chromosome DSD, and its diagnosis is mainly based on cytogenetic or molecular detection of XX/XY leukocyte chimerism [1][2][3]. Other forms of sex chromosome DSD, such as sex chromosome aneuploidies, have rarely been reported in cattle, while there have been no reports of gene mutations responsible for the DSD phenotype in individuals with the normal complement of sex chromosomes-i.e., XX DSD or XY DSD [4].
The identification of the mechanisms causing DSD phenotype is an important issue from the point of view of breeding. Some DSDs have a de novo origin (e.g., freemartinism, X monosomy, and XXY syndrome) and are not heritable, as the affected animals are sterile. On the contrary, carriers of gene mutations responsible for XX DSD or XY DSD can easily spread the mutation in populations. It is important to point out that distinguishing between heritable and non-heritable DSDs, based on the appearance of external genitalia, is not possible.
In domestic animals, heritable forms of XX DSD are quite common, but it is associated with the presence of ovotestis or testis. Until now, the causative mutation affecting the expression of the FOXL2 gene involved in ovarian development has been identified only in goats [5,6]. In pigs, the XX DSD is associated with DNA variants in a region harboring the SOX9 gene, which plays a crucial role in development of the testes [7,8]; in dogs, it is associated with variants near SOX9 or PADI6 [9][10][11][12]. Upstream DNA variants of SOX9 are also known to cause of XX DSD in humans [13].
In cattle, three cases of XX DSD have been reported, and in all these cases, sequences derived from the Y chromosome were detected in the urogenital organs [14], leukocytes [15], blood cells, ovaries, and lymph nodes [16]. However, the SRY gene has only been detected in two reports [14,16]. Interestingly, in some DSD heifers, mosaicism with the presence of a triploid cell line carrying the Y chromosome (60,XX/90,XXY) has also been observed (summarized in [17]).
In this study, we analyzed five DSD heifers with ambiguous external genitalia, including a case with extensive virilization. This case was the main subject of molecular analysis due to the presence of a normal set of female sex chromosomes (60,XX) in leukocytes.

Animals
Five heifers (four Holstein Friesians and one Limousin × Simmental crossbred) were subjected to genetic analysis on the request of breeders or veterinarians due to the presence of ambiguous external genitalia (Table 1). These heifers were not related and originated from four farms located in western or central Poland. In four of the heifers, enlarged clitoris or extended anus-vulva distance was observed (Figure 1a-d). The most extensive virilization was observed in the fifth case (#7514), with a rudimentary vulva being ventrally located near mammary gland (Figure 1e,f).

Histological Studies
Samples of the uterus (approx. 4 cm), oviducts (approx. 1.5 cm), and gonads (approx. 3 and 4 cm) collected postmortem were fixed in neutral buffered 10% formalin solution and used for preparation of paraffin sections (3 µm), which were stained with hematoxylin and eosin (H&E). Microscopic observations were carried out under an Axio Lab.A1 microscope (Carl Zeiss, Oberkochen, Germany) equipped an ERc5s digital camera (Carl Zeiss, Oberkochen, Germany) and analyzed with the use of Zen 2.3 software (blue edition; Carl Zeiss Microscopy, 2011).

Cytogenetic Analysis
Blood samples were collected in heparinized tubes for establishing short-term (48 h) in vitro leukocyte cultures. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15% (v/v) fetal calf serum and 1% (v/v) penicillin/streptomycin and phytohemagglutinin at 37 • C in a humidified atmosphere of 5% CO 2 . A standard cell culture harvesting procedure was used, including colcemid, hypotonic and fixative treatments. Chromosomes were analyzed using Giemsa staining and C-and Gbanding techniques (applied to case #7514), according to methods reviewed by Iannuzzi and Di Berardino [18]. Bovine sex chromosomes were identified based on their biarmed morphology (a large submetacentric X and a small metacentric Y), contrasting with the one-arm morphology of all autosomes, lack of centromeric C band and the characteristic G banding pattern (case #7514). One hundred metaphase Giemsa-stained spreads were analyzed for each case. In addition, twenty C-banded and G-banded spreads derived from DSD heifer #7514 were also evaluated. The slides were examined with an epifluorescence Nikon E600 Eclipse microscope (Melville, NY, USA) equipped with a cooled CCD digital camera (Melville, NY, USA) and Lucia software (Laboratory Imaging, Prague, Czech Republic).

Molecular Detection of X-Linked and Y-Linked Genes
DNA was isolated from blood using DNA Blood Mini kit (A&A Biotechnology, Gdansk. Poland) and from hair follicles using Sherlock AX kit (A&A Biotechnology, Gdansk, Poland). The SRY gene fragment covering the whole coding sequence (851 bp) was amplified by PCR using the primers shown in Supplementary Table S1, and its presence was verified using agarose gel electrophoresis. The X-linked and Y-linked (ZFX and ZFY, respectively) genes were amplified (448 bp) by PCR (Supplementary Table S1) and distinguished by restriction enzyme (BsmI) digestion at 37 • C for 4 h following agarose gel electrophoresis (448 bp for ZFY; 391 and 57 bp for ZFX). Moreover, PCR detection of the Y-chromosome-derived genes was also performed on DNA samples isolated from the ovaries, uterus, and oviduct (Genomic Mini kit, A&A Biotechnology, Gdansk). All PCR primers were designed using Primer3 (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; accessed on 10 August 2009), and all details (primer sequences, annealing temperatures and the amplicon lengths) are shown in Supplementary Table S1.

Analysis of SOX9 and AMELY/AMELX Copy Number
Droplet digital PCR (ddPCR) was used to detect the AMELX (X-linked) and AMELY (Ylinked) genes, with a fluorescent ratio of AMELY/AMELX amplicons below 1.0 confirming the presence of XX/XY chimerism, following the procedure described by Szczerbal et al. [3]. Moreover, ddPCR was also used to estimate the copy number of the SOX9 gene by taking the copy number of the F2 autosomal gene as a reference [19]. To establish the amplicon ratio of the Y-derived gene (AMELY) to the autosomal gene (SOX9), an additional reaction was performed with these genes. The procedure described by Nowacka-Woszuk et al. [11] was followed. Briefly, the reaction mixture contained 10 µL of 2×ddPCR Supermix for Probes (Bio-Rad, Hercules, CA, USA), 1 µL of 20× primers/FAM probe, 1 µL of 20× primers/HEX probe, and 1 µL of the BsuI and HaeIII restriction enzymes for the AMELX and AMELY genes and the SOX9 and F2 genes, respectively. The PCR mixtures were partitioned into approximately 20,000 droplets using a QX200 droplet generator (Bio-Rad, Hercules, CA, USA). PCR was run using following conditions: denaturation at 95 • C for 10 min; 40 cycles at 94 • C for 30 s, and at 55 • C (for AMELX and AMELY), 57 • C (for SOX9 and F2) and 56 • C (for AMELY and SOX9) for 60 s (ramp rate 2 • C/s); 98 • C for 10 min, and 10 • C until reading time. The droplets were analyzed on a QX200 droplet reader (Bio-Rad, Hercules, CA, USA). The concentration of the genes was calculated by Poisson distribution using Quantasoft software (Bio-Rad, Hercules, CA, USA). The primer and probe sequences are shown in Supplementary Table S1.

Genotyping of Selected Tissues Using Microsatellite Markers
The genotyping of DSD heifer #7514 was performed using microsatellite (short tandem repeats-STR) markers in DNA samples collected from the blood, hair follicles, ovaries, uterus, and oviduct. Altogether, twelve markers (BM1818, BM1824, BM2113, ETH3, ETH10, ETH225, INRA23, SPS115, TGLA53, TGLA122, TGLA126 and TGLA227) recommended by the International Society of Animal Genetics (ISAG) for parentage testing and genetic profiling were analyzed by a certified laboratory at the Institute of Animal Production (Balice, Poland). Briefly, the analysis was based on the amplified fragment length polymorphisms (AFLP) method, where all markers were amplified in a single multiplex using TypeIt Microsatellite PCR Kit (Qiagen, Hilden, Germany). The amplicons were separated by capillary electrophoresis on Genetic Analyzer 3500 xL (Applied Biosystems, Waltham, MA, USA) with the use of POP-7 polymer (Thermofisher Scientific, Waltham, MA, USA). The length of amplicon was determined using GeneMapper Software 5 (Applied Biosystems, Waltham, MA, USA).

Results
The microscopic evaluation of cytogenetic slides obtained from in vitro cultured leukocytes could be performed for the four DSD heifers (#7502, #7514, #7515 and #7518), and the molecular detection of the Y-derived sequences could be performed for all DSD heifers (#7497, #7502, #7514, #7515 and #7518).
A normal XX sex chromosome complement, analyzed by Giemsa staining, as well as by C-and G-banding (Figure 2) was observed in one of the heifers (#7514), and XX/XY leukocyte chimerism was detected in the other three (#7502, #7515, and #7518) (data not shown).  Figure S1). In addition, the presence of the chimerism was confirmed by estimating the Y/X copy number through ddPCR, based on the number of amplicons derived from the AMELY (Y-derived) and AMELX (X-linked) genes (Supplementary Figure S2). The results were concordant with those of the cytogenetic analysis. On the basis of these results, case #7514 was tentatively classified as an XX (SRY-negative) DSD, while the remaining four cases appeared to be typical freemartins (Table 1). Further analysis focused on DSD heifer #7514. Postmortem anatomical examination of the genitourinary system revealed normal female internal genitalia and virilized external genital organs. The uterine horns and cervix were of normal structure, shape, and consistency (Figure 3a). The ovaries were also normal in structure and shape but contained only a few follicles and corpora lutea. Both oviducts were complete and normal in size. The vagina, despite its normal structure in the cranial part, was dilated in the caudal part. Vulva, vestibule, cervix, uterine body and uterine horns were connected to each other and unobstructed. The absence of a vulval cleft in the perineal area was noted. The urinary bladder and ureter were of normal structure and shape. The urethral orifice was in its normal position in the vagina, and the urethra was connected to the bladder. There was a hypoplastic penile-like structure with a penile retractor muscle connected to the vestibule of the vagina; inside this, there was a virilized urethra with a secondary external orifice located on the ventral body aspect, cranial to the udder. This was in the form of a vulval cleft-like structure or preputial-like structure. The male external genitalia were absent from the inguinal area.
Histological analysis of gonads, oviducts, and uterus showed them to have normal structure (Figure 3b-d). In the ovaries were observed follicles, including a Graafian follicle, as well as corpora lutea and corpora albicans. No structures resembling testicular organization were found.
We focused in the first step of the molecular study on elucidating the background of the observed phenotype in DSD heifer #7514. PCR revealed the presence of Y-chromosomederived genes (SRY and ZFY) in the gonads, oviduct, and uterus ( Figure 4). This observation indicated the presence of another cell line or lines.  We thus genotyped DNA samples isolated from blood, hair follicles, ovaries, oviduct, and uterus at twelve microsatellite loci, as is commonly done in parentage testing. Additional variants were observed at six loci (ETH3, ETH10, ETH225, SPS115, TGLA53, and TGLA227) in the internal genitalia, while only one or two variants were found in blood and hair follicles ( Figure 5; Supplementary Table S2). This result, indicating the presence of chimerism in the internal genitalia, was followed by molecular detection of the number of copies of sex chromosomes and autosomes. Firstly, ddPCR was used to estimate the number of copies of the SOX9 gene, since an elevated number usually affects gonadal development in females (ovotestis or testis instead of ovaries) and causes virilization. In all studied tissues, the copy number of SOX9 was normal (two copies), as it was also observed in the studied freemartins (Supplementary Figure S3). Next, ddPCR was used to estimate the Y/X ratio, on the basis of the amplicon numbers of the AMELY (Y-linked) and AMELX (X-linked) genes. The expected Y/X ratio for a normal male cell line carrying X and Y chromosomes is 1.0, while for a normal female line (XX), it is 0. In our case, the Y/X ratio was low (<0.3, Figure 6), thus confirming the presence of two cell lines (XX and XY). In addition, the AMELY/SOX9 ratio was lowered than the expected 0.5 for a single XY cell line (Supplementary Figure S4). This also indicated the presence of another cell line carrying the Y chromosome in this heifer.

Discussion
The incidence, consequences, and background of bovine freemartinism have been frequently reported on Esteves et al. [2]. It is well-known that such heifers have underdeveloped internal genitalia and that their external genitalia are often virilized. Heifers born as co-twins to males are usually culled due to the high risk of freemartinism (>90%). However, some freemartins are born as singletons due to early fetal death of the male co-twin [20]. Where virilized genitalia are observed in such heifers, distinguishing between nonheritable freemartinism and other types of DSD requires genetic analysis. Unfortunately, knowledge of the mechanisms responsible for DSD phenotype in heifers with a normal set of XX chromosomes is scarce.
To our best knowledge, there have only been three reports of XX DSD heifers, and in all these cases, Y-chromosome-derived sequences were detected [14][15][16]. In the heifer reported by Takagi et al. [14], a vulval orifice-like structure localized ventrally and cranially to the mammary gland, as well as normal internal genitalia, including ovaries with follicles and corpora lutea, were observed. Interestingly, DSD case #7514 in the present study had a very similar phenotype, and in both cases three Y-linked genes (SRY, ZFY and AMELY) were detected in the internal genitalia, though not in blood cells.
A different XX DSD heifer phenotype was reported by Payan-Carreira et al. [15], who observed rudimentary external genitalia with a small clitoris-like structure, bilateral streak gonads, a normal uterus, a long vagina, and the urethral orifice at the normal location. Fluorescent in situ hybridization (FISH) with a genomic degenerate oligonucleotide-primed (DOP)-PCR probe derived from the heifer revealed the presence of Y chromosome sequences in both X chromosomes, though the SRY gene was not detected by PCR.
The third XX DSD case reported in the literature was a female with a normal uterus, ovaries, and mammary gland, but also with a prepuce, normally urinating penis, and scrotum [16]. Molecular analysis revealed the presence of the SRY gene in several tissues, including the blood, ovaries, and lymph nodes.
The external genitalia of the DSD heifer (case # 7514) described here were extensively virilized, though a normal uterus and ovaries with follicles were observed. Since earlier reports of XX DSD heifers indicated the presence of Y-derived sequences in some organs, we also searched for three Y-derived genes (SRY, ZFY and AMELY); we detected them in internal genitalia. Our study thus confirmed that the presence of Y-chromosome-derived genes plays a crucial role in the virilization of XX DSD heifers with female internal genitalia, including ovaries.
It is well-known that the presence of a functional SRY gene triggers the development of undifferentiated fetal gonads of mammals toward testis, while ovaries develop when functional SRY transcription factor is not expressed [21]. Thus, the detection of the SRY gene in ovaries is a very unusual situation. On the other hand, there are reports suggesting that the XX/XY chimerism can be present in both blood cells and gonadal tissue of bulls originating from heterosexual twins [22,23]. There has also been a report concerning an XY (SRY-positive) DSD heifer, in which the SRY gene was detected in the blood and ovaries with follicles and a large corpus luteum, despite XX/XY chimerism (freemartinism) being excluded though analysis of in vitro cultured leukocytes and genotyping of eighteen microsatellite markers in DNA isolated from blood [24]. In the present case, we also observed a normal set of female sex chromosomes in leukocytes, and the analysis of microsatellites in DNA isolated from blood cells excluded the presence of the chimerism. In fact, it was in the internal genitalia that the chimerism was detected, as it was revealed by the microsatellite genotyping and ddPCR of sex-linked genes.

Conclusions
Our study has shown the presence of XX/XY chimerism in internal genitalia, including ovaries, in a DSD heifer with a normal set of female sex chromosomes (XX) in the leukocytes. Since chimersism was not observed in leukocytes, we could exclude a classical form of freemartinism. The most intriguing issue of the presence of the SRY gene in normally developed ovaries and the mechanisms responsible for the migration of XY cells to internal female genitalia requires further studies to be elucidated.

Informed Consent Statement:
The blood samples were collected by veterinarians with the consent of heifers owners for reporting the obtained scientific results.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.