**Comparative Fluorescence In Situ Hybridization (FISH) Mapping of Twenty-Three Endogenous Jaagsiekte Sheep Retrovirus (enJSRVs) in Sheep (***Ovis aries***) and River Buffalo (***Bubalus bubalis***) Chromosomes**

**Angela Perucatti 1, Alessandra Iannuzzi 1,\*, Alessia Armezzani 2, Massimo Palmarini <sup>2</sup> and Leopoldo Iannuzzi <sup>1</sup>**


**Simple Summary:** The genome of domestic sheep (*Ovis aries*) harbors at least twenty-seven copies of enJSRVs, endogenous retroviruses (ERVs) highly related to the exogenous and pathogenic Jaagsiekte sheep betaretrovirus (JSRV). Interestingly, some of these loci are insertionally polymorphic, that is they are present only in some individuals or populations of their host species. This differential distribution of enJSRVs has provided important insights into tracing host and viral evolution. In this study, we report the first comparative fluorescent in situ hybridization (FISH) mapping of genetically characterized enJSRVs in domestic sheep (2n = 54) and river buffalo (*Bubalus bubalis*, 2n = 50), and reveal a high conservation of enJSRVs chromosome localization between these two species.

**Abstract:** Endogenous retroviruses (ERVs) are the remnants of ancient infections of host germline cells, thus representing key tools to study host and viral evolution. Homologous ERV sequences often map at the same genomic locus of different species, indicating that retroviral integration occurred in the genomes of the common ancestors of those species. The genome of domestic sheep (*Ovis aries*) harbors at least twenty-seven copies of ERVs related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRVs), thus referred to as enJSRVs. Some of these loci are unequally distributed between breeds and individuals of the host species due to polymorphic insertions, thereby representing invaluable tools to trace the evolutionary dynamics of virus populations within their hosts. In this study, we extend the cytogenetic physical maps of sheep and river buffalo by performing fluorescent in situ hybridization (FISH) mapping of twenty-three genetically characterized enJSRVs. Additionally, we report the first comparative FISH mapping of enJSRVs in domestic sheep (2n = 54) and river buffalo (*Bubalus bubalis*, 2n = 50). Finally, we demonstrate that enJSRV loci are conserved in the homologous chromosomes and chromosome bands of both species. Altogether, our results support the hypothesis that enJSRVs were present in the genomes of both species before they differentiated within the *Bovidae* family.

**Keywords:** sheep; river buffalo; endogenous retrovirus; FISH-mapping; cytogenetic map

#### **1. Introduction**

Retroviruses possess the unique ability to integrate into the genome of infected cells. Occasionally, they can infect germline cells and give rise to endogenous retroviruses (ERVs), retroviral sequences transmitted vertically, in a Mendelian fashion, as part of the host genome. As such, ERVs represent fascinating tools to study both virus and host genome evolution [1]. ERVs have been found in all vertebrates studied to date, including fish, amphibians, birds, reptiles, and mammals [2]. Comparative genomic studies have shown that related species often share ERV families or specific ERV loci, and that, in many

**Citation:** Perucatti, A.; Iannuzzi, A.; Armezzani, A.; Palmarini, M.; Iannuzzi, L. Comparative Fluorescence In Situ Hybridization (FISH) Mapping of Twenty-Three Endogenous Jaagsiekte Sheep Retrovirus (enJSRVs) in Sheep (*Ovis aries*) and River Buffalo (*Bubalus bubalis*) Chromosomes. *Animals* **2022**, *12*, 2834. https://doi.org/10.3390/ ani12202834

Academic Editor: Ettore Olmo

Received: 27 August 2022 Accepted: 17 October 2022 Published: 19 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cases, homologous ERV sequences map at the same genomic locus in multiple species' genomes, indicating that retroviral integration events occurred in the genomes of the common ancestors of those species [3,4].

In 2013, Garcia-Etxebarria and Jugo traced the evolutionary history of bovine ERVs (BoERVs) by performing computational analyses on the genomes of several bovid species, including cattle, sheep, goat, and water buffalo [5]. Interestingly, they found twenty-six BoERV families in all the species studied, suggesting that most of these families could be present in all members of the *Bovidae* family. However, they could not detect four BoERV families (i.e., BoERV24, BoERV26, BoERV28, and BoERV29) in sheep or goat genomes, indicating that these families may be specific to the *Bovinae* subfamily. The authors hypothesize that the majority of the BoERV families invaded the genome of the common ancestor of the *Bovidae* family approximately 20 million years ago (MYA) and only later, between 12MYA and 20MYA, the ancestors of the BoERV24, BoERV26, BoERV28, and BoERV29 families might have been inserted into the genome of the *Bovinae* subfamily ancestor [5].

The domestic sheep (*Ovis aries*) harbors at least twenty-seven ERV loci related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV), thus referred to as enJSRVs [6,7]. Interestingly, some enJSRV loci are insertionally polymorphic, that is they are present only in some individuals or populations of their host species. This differential distribution has provided important insights into tracing the evolutionary dynamics of virus populations within their hosts [8].

Along these lines, in previous studies, we used enJSRVs to (i) investigate the history of sheep domestication [9], (ii) explore the molecular mechanisms through which the most recent enJSRV—enJSRV-26—eludes the restriction exerted by enJS56A1 (which entered the sheep genome before and during the speciation within the genus *Ovis*) [10], or (iii) study the chromosome location of some enJSRV loci by fluorescent in situ hybridization (FISH) analyses of metaphase R-banded sheep chromosomes [9,10].

In the present work, we extend the cytogenetic physical maps of both sheep and river buffalo chromosomes by FISH mapping fifteen and twenty-three additional enJSRVs, respectively. In addition, we perform the first comparative FISH mapping of genetically characterized enJSRVs in domestic sheep and river buffalo (*Bubalus bubalis*).

#### **2. Materials and Methods**

*Cell cultures.* Peripheral blood lymphocytes of sheep (*Ovis aries*, OAR; 2n = 54) (four animals) and river buffalo (*Bubalus bubalis*, BBU; 2n = 50) (three animals) were cultured for 72 h in Roswell Park Memorial Institute (RPMI)-1640 culture medium enriched with 15% fetal calf serum (FCS), concanavalin A (15 μg/mL), penicillin/streptomycin (0.1 mL), L-glutamine (0.1 mL), and one drop of sterile sodium heparin to prevent coagulation. In order to obtain R-banding patterns and cause metaphase arrest, 7 h prior to harvesting, cells were labeled with BrdU (15 μg/mL) and Hoechst 33258 (30 μg/mL) and, 6 h later, they were treated with colcemid (0.1 μg/mL). After incubation in a hypotonic solution (KCl 0.075 M at 37.5 ◦C for 20 min), cells were fixed three times with 3:1 methanol–acetic acid (*v*/*v*) solution. Cell suspensions were then spread onto a slide, and stored at −20 ◦C.

*Probes and FISH mapping.* FISH analysis was performed using standard procedures [11,12]. Briefly, cells were pre-treated overnight at 50 ◦C, and subsequently stained with Hoechst 33258 (25 μg/mL) for 10 min. Slides were then exposed to UV light for 20 min, washed with distilled water, and air-dried. Hybridization, chromosome staining, signal detection and image processing were carried out as already described [11,12]. Slides were mounted in antifade mounting medium with propidium iodide to visualize both FITC-signals and RBPI-banding using two microscope filter combinations. Two images for each metaphase were acquired with both FITC signals and RBPI-banding. Next, FITC signals were superimposed on RBPI-banding to get a precise position of FITC-signals on chromosome bands. Thirty metaphases for each probe were examined. Chromosome identification and banding numbering system followed the standard nomenclature of both species [13,14]. The bacterial artificial chromosome (BAC) clones used for sheep cytogenetic mapping have been already characterized and described

elsewhere [6]. Notably, each BAC clone used to obtain the sequences of the various enJSRV loci was subjected to Southern blot analyses to verify that it contained only one locus. The list of sheep BAC clones of the CHORI-243 library containing the 27 enJSRVs used in this study is reported in Table 1:


**Table 1.** List of sheep BAC clones containing the 27 enJSRVs used in the present study.

#### **3. Results**

In the present study, we conducted FISH mapping on sheep and river buffalo chromosomes (or chromosome arms) using twenty-seven sheep BAC clones (Table 1). As reported in Table 2, we obtained good hybridization signals only with twenty-three BAC clones, allowing us to map the corresponding twenty-three enJSRV loci. Interestingly, we localized these loci on twelve different chromosomes (or chromosome arms) of both species. As reported in Table 1, we found that, for all the probes used, hybridization signals and chromosome bands localized in the same homologous chromosome pairs of both sheep and river buffalo (Table 2). For example, we found that the enJSRV-1 and enJSRV-10 loci map onto homologous sheep and river buffalo R-banded chromosomes, as shown in Figure 1. Moreover, the BAC containing the enJSRV-1 map onto two different chromosomes in both sheep (chromosomes 6 and 18) and river buffalo (chromosomes 7 and 20) (Figure 1 and Table 2). In addition, the enJSRV-15, enJSRV-20, enJSRV-21, and enJSRV-27 display the same chromosome localization as the enJSRV-1 in both species (OAR6q13 and BBU7q21, respectively) (Table 2). Finally, the enJSRV-2 and enJSRV-6 map very close in both sheep (OAR1q45 and OARiq43, respectively) and river buffalo homologous chromosomes (BBU1q45 and BBU1q43, respectively). Notably, only the enJSRV-7 maps onto the centromeric regions of all autosomes [10], probably due to the presence of highly repetitive sequences in the BAC clones, the centromeric regions being highly rich in these sequences.


**Table 2.** Chromosomal localization of 23 enJSRVs in sheep (OAR) and river buffalo (BBU) chromosomes by FISH-mapping.

**Figure 1.** FISH mapping of enJSRV-1 (**A**,**B**) and enJSRV-10 (**C**,**D**) in sheep (OAR) and river buffalo (BBU) chromosomes. Two different images were taken, with hybridization FITC signal and with RBPI-banding. Subsequently, hybridization FITC signals were superimposed on RBPI-banding to get a precise localization of mapped loci in both species. Note that enJSRV-1 maps onto two different chromosomes in both species (**A**,**B**).

#### **4. Discussion**

FISH represents a very powerful cytogenetic technique for mapping a particular genomic sequence on a chromosome [15,16], and better anchoring of radiation hybrid (RH) and genomic maps to specific chromosome regions [17–20]. In more recent years, FISH has also been recognized as a reliable diagnostic and discovery tool to evaluate genetic anomalies, by studying chromosomal aberrations in both metaphase and interphase nuclei [reviewed in [21–25], and defects in chromosome segregation during meiosis [26,27]. In addition, implementations of FISH with whole-chromosome painting have led to the generation of detailed comparative maps to study chromosomal homologies and divergences between related and unrelated species [28–32]. Finally, FISH has also become instrumental in generating detailed comparative maps to study gene order, conserved chromosomal regions, and chromosomal rearrangements between related and unrelated species [33–36].

Along these lines, FISH analyses unveiled the phylogenetic relationships between the *Caprinae* subfamily and the earliest-diverging *Bovinae* subfamily, by showing two main chromosome events occurring at the autosomes 9 and 14, and the sex chromosomes (mainly the X-chromosome). More specifically, in previous studies carried out in our group, we demonstrated that a chromosome transposition has occurred from the proximal region of *Bovinae* chromosome 9 to the proximal region of *Caprinae* chromosome 14, and that at least four chromosome rearrangements (i.e., three transpositions and one inversion) differentiated the *Caprinae* from the *Bovinae* X-chromosomes reviewed in [37].

A comparative FISH mapping of enJSRVs has been reported previously in sheep and goat cell lines [38]. In this study, the authors only partially found enJSRVs localized on the same homologous chromosome band of the two species. In addition, they found enJSRV loci in seven and eight chromosomes of sheep and goats, respectively.

In the present work, we report the first comparative FISH mapping between two species belonging to the *Caprinae* (*Ovis aries*) and *Bovinae* (*Bubalus bubalis*) subfamilies of the *Bovidae* family by using well-identified and genetically characterized enJSRVs [5,6]. We show that hybridization signals of enJSRVs are found in at least twelve different chromosomes (or chromosome arms) of both species, and that all mapped loci are conserved in homologous chromosome regions and chromosome bands of these two species (Table 1). However, since BACs contain large genomic inserts, it is entirely possible that, besides enJSRVs, we also simultaneously mapped other genes and sequences present on such clones that share homology with some river buffalo chromosomal regions. Indeed, since some BoERV families (i.e., BoERV24, BoERV26, BoERV28, and BoERV29) are specific to the *Bovinae* family [5], the same could have occurred for some enJSRV which are present in the *Caprinae* subfamily (i.e., *Ovis aries*) but not in the *Bovinae* subfamily (i.e., *Bubabus bubalis*).

We observed that, in both species, the BAC clones containing enJSRV-1 map onto two different chromosomal locations (Table 2). Interestingly, we found that enJSRV-7 is the only locus mapping at the centromeric regions of both sheep [10] and buffalo chromosomes.

The same results were achieved by FISH mapping several ERV in sheep chromosomes which exhibited abundant centromeric to the dispersed distribution of various endoviruses, probably due to the abundance of genomic organization ERV-related repetitive elements which are particularly present at the centromeric regions of the chromosomes [39].

Our comparative FISH mapping in two different bovid species further confirms the high degree of chromosome (and chromosome arm) conservation among bovids reviewed in [37]. In addition, our study supports the hypothesis that enJSRVs were present in the genomes of their bovine ancestor before the differentiation of the *Caprinae* subfamily (including *Ovis aries*) from the most ancient *Bovinae* subfamily (including *Bubalus bubalis*) [37,40]. These results are in agreement with those published in a previous study [5] tracing BoERVs evolution in several species of the *Bovidae* family, including cattle, sheep, goats, and water buffalo [5]. Interestingly, these authors found that most of the BoERV families are present in all the species studied, supporting the hypothesis that BoERVs entered the genome of the common ancestor of the *Bovidae* family about 20 MYA or less. In addition, they detected higher BoERV copy numbers

in cattle compared to other bovid species, suggesting that an additional expansion of retroviral copies might have occurred in the cattle genome [5].

Interestingly, we found five enJSRV loci (enJSRV-1, enJSRV-15, enJSRV-20, enJSRV-21, and enJSRV-27) on the same chromosome band (OAR6q13/BBU7q21) (Table 2). Previous FISH mapping conducted in our group revealed two type-one loci on this very same chromosome band: the pyroglutamylated RFamide peptide receptor (QRFPR) and the translocation-associated membrane protein 1-like 1 (TRAM1L1) [41]. The RFamide peptide family consists of several groups, including the neuropeptide FF group, the prolactinreleasing peptide group, the gonadotropin inhibitory hormone group, the kisspeptin group, and the pyroglutamylated RFamide peptide (26RFa/QRFP) group [42]. Interestingly, pyroglutamylated RFamide peptide 43 has been proven to be a putative modulator of testicular steroidogenesis, playing an important role in reproduction [43]. Notably, ERVs are key in placental morphogenesis and mammalian reproduction [44]. TRAM1L1 seems to be closely related to chronic widespread musculoskeletal pain (CWP), a common disorder affecting about 5–15% of the population, and one of the main symptoms of fibromyalgia, which has been shown to be associated with an altered gut microbiome [45]. By using the sheep genome reference sequence (https://www.ncbi.nlm.nih.gov/genome/ gdv/browser/genome/?id=GCF\_016772045.1, accessed on 12 May 2018), we identified sixteen genes included between QRFPR and TRAM1L (Supplementary Table S1). In this table we reported some of the functions of those genes (including QRFPR and TRAM1L), mostly involved in anti-tumor immune response probably to counteract the presence of several enJSRVs in these chromosomic regions. Indeed, genomic amplification within the 6q13 region was detected, and it was found that the number of enJSRV-6q13 is correlated to the number of protective mutations [46].

#### **5. Conclusions**

To our knowledge, this is the first comparative FISH mapping of sheep and river buffalo chromosomes using genetically characterized enJSRVs. Interestingly, our results reveal a high degree of conservation of enJSRVs localization in the homologous chromosomes and chromosome bands of both species. These findings support the hypothesis that enJSRVs entered the host genome before the differentiation of the *Caprinae* subfamily from the earliest-diverging *Bovinae* subfamily of the *Bovidae* family. Finally, the present study extends the current genetic physical maps of sheep and river buffalo by mapping, respectively, fifteen and twenty-three additional enJSRV loci on the chromosomes and chromosome arms of these two species.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani12202834/s1, Table S1: List of genes present in the ORA6q13 and included between QRFPR and TRAM1L1 (from: https://www.ncbi.nlm.nih.gov/genome/gdv/browser/ genome/?id=GCF\_016772045.1), accessed on 2 October 2022) their main functions and references.

**Author Contributions:** Conceptualization L.I. and M.P.; investigation, A.P. and A.A.; data curation, A.P. and A.I.; writing—original draft preparation L.I., A.A. and A.I.; writing—review and editing, A.I., L.I., A.P., A.A. and M.P.; supervision, L.I. and M.P.; funding acquisition L.I. and M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the project PON1\_486 GENOBU.

**Institutional Review Board Statement:** The study was approved by the CNR-ISPAAM Ethics Committee, protocol number 0000606 of 03/27/2015.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author, [AI], upon reasonable request.

**Acknowledgments:** We are grateful to Domenico Incarnato, CNR-ISPAAM, Portici, Italy for his excellent technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **XX/XY Chimerism in Internal Genitalia of a Virilized Heifer**

**Izabela Szczerbal 1,†, Joanna Nowacka-Woszuk 1,†, Monika Stachowiak 1, Anna Lukomska 2, Kacper Konieczny 3, Natalia Tarnogrodzka 1, Jakub Wozniak <sup>1</sup> and Marek Switonski 1,\***


**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 Ychromosome-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.

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

**Citation:** Szczerbal, I.; Nowacka-Woszuk, J.; Stachowiak, M.; Lukomska, A.; Konieczny, K.; Tarnogrodzka, N.; Wozniak, J.; Switonski, M. XX/XY Chimerism in Internal Genitalia of a Virilized Heifer. *Animals* **2022**, *12*, 2932.

Academic Editors: Leopoldo Iannuzzi and Pietro Parma

https://doi.org/10.3390/ ani12212932

Received: 14 September 2022 Accepted: 24 October 2022 Published: 26 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. 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 [1–3]. 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* [9–12]. 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.

#### **2. Material and Methods**

#### *2.1. 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).

**Figure 1.** Virilized external genitalia of studied cases. (**a**) Case #7497. (**b**) Case #7502. (**c**) Case #7515. (**d**) Case #7518. (**e**,**f**) Case #7514.




**Table 1.** *Cont.*

\* HF: Holstein Friesian; L × S: Limousin × Simmental crossbred.

#### *2.2. 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).

#### *2.3. 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% CO2. 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).

#### *2.4. 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 (*Bsm*I) 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.

#### *2.5. 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 *Bsu*I and *Hae*III 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.

#### *2.6. 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).

#### **3. 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). The proportion of XX and XY metaphase spreads varied from XX [98%]/XY [2%] to XX [25%]/XY [75%] (Table 1). In the next step, Y-derived genes (*SRY* and *ZFY*) were not detected in case #7514. On the other hand, both genes were found in blood cells, though not in DNA samples isolated from hair follicles, in the other four cases (#7497, #7502, #7515 and #7518) (Supplementary 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).

**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.

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.

**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).

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).

**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.

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.

**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).

#### **4. 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 [14–16]. 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.

#### **5. 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.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ani12212932/s1. Supplementary Table S1. PCR conditions applied in molecular analysis of the selected genes. Supplementary Table S2. STR markers analyzed in DSD heifer #7514. Supplementary Figure S1. Detection of *SRY* (a) and *ZFY* (b) genes in freemartin cases. L: GeneRuler DNA ladder; B: blood; H: hair follicles; M: control male; FM: control female; NC: negative control (no DNA). Supplementary Figure S2. Estimation of the Y/X ratio by ddPCR based on the proportion of *AMELY* and *AMELX* amplicons in freemartin cases. (a) amplification signals from chromosome X (blue color) and Y (green color). (b) Y/X ratio is presented. B: blood; H: hair follicles; M: control male; FM: control female; NC: negative control (no DNA). Supplementary Figure S3. *SOX9* copy number by ddPCR for freemartin cases (a) and DSD heifer #7514 (b). B: blood; H: hair follicles; O1: ovary 1; O2: ovary 2; U: uterus; OD: oviduct; FM: control female; NC: negative control (no DNA). Supplementary Figure S4. Estimation of the Y/autosome ratio by ddPCR based on the proportion of *AMELY* and *SOX9* amplicons in the DSD heifer #7514. 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).

**Author Contributions:** Each author made substantial contributions to this project. I.S. performed cytogenetic studies and contributed to molecular studies and preparation of the manuscript. J.N.-W. performed molecular studies and contributed to the preparation of the manuscript. M.S. (Monika Stachowiak) performed molecular studies. A.L. performed histological studies. K.K. performed anatomical studies and contributed to the collection of the samples. N.T. contributed to cytogenetic studies and the collection of the samples. J.W. contributed to molecular studies and the collection of the samples. M.S. (Marek Switonski) designed and supervised the study, prepared the manuscript and contributed to the collection of the samples. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was financed from the statutory funds of the Department of Genetics and Animal Breeding (No. 506.534.05.00), Faculty of Veterinary Medicine and Animal Science, Poznan University of Life Sciences, Poznan, Poland.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the local Bioethical Commission for Animal Care and Use in Poznan, Poland (certificate no 3/2019).

**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.

**Acknowledgments:** We thank veterinarian Konstancja Balcer for blood sample collection.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**

