Early Diagnosis of 60, XX/XY Chimerism in Cattle using RT-PCR

One of the most common kinds of intersexuality found in cattle is freemartinism (Padula, 2005; Di Meo et al., 2008; Peretti et al., 2008), a sterile female calf, born with a male co-twin healthy fetus, with rudimentary or undeveloped genitalia (Esteves et al., 2012). Usually, freemartinism occurs in early stages of development due to formation of vascular anastamosis between heterosexual fetuses, which ultimately leads to disrupted differentiation of female gonads and genital tract in further developmental stages (Kozubska-Sobocinska et al., 2016, Padula, 2005; Schlafer and Miller, 2007). The male co-twin shows only minimal defects with slight reduction in fertility (Padula, 2005).

Earlier reports, revealed that 82 to 97 per cent of female calves born co-twin with males showed 60, XX/60, XY leukocyte chimerism (Slota et al., 2004; Padula, 2005; Esteves et al., 2012; Qiu et al., 2018), which, will be sold for meat or production of fetal bovine serum (Hirayama et al., 2007). However, there were reports of fertile female co-twins (Qiu et al., 2018; Bierman et al., 2010 and Szczerbal et al., 2014). Hence, early diagnosis of female calves of heterosexual pregnancies would prevent dispensable economic loss and preserve valuable genetic material (Biswas et al., 2015). Hence, the objective of the present study was to investigate the suitability of female and male co-twins for further breeding, at early age by screening through cytogenetic and molecular genetic techniques.

Clinical case
 
The samples were collected from the Large animal gynecology ward, Madras Veterinary College, Chennai. The work has been carried out between august, 2019 to December, 2019. Heterosexual twin calves (10 days) born to an indigenous (non-descript) cow were presented with female calf having abnormal external genetalia with long coarse of hairs at ventral commissure (Fig 1). Blood samples were collected from both male and female calves in sodium heparin vials and transported to laboratory at 4°C for cytogenetic and molecular studies.


 
Cytogenetic analysis
 
Chromosomes were analyzed using short term lymphocyte culture technique standardized in Cytogenetics Laboratory, Madras Veterinary College, Chennai (Harshini et al., 2020). A minimum of 200 metaphase spreads per animal were examined to estimate the proportion of 60, XX and 60, XY cells.
 
Molecular analysis
 
Genomic DNA was isolated using standard phenol-chloroform extraction procedure (Sambrook et al., 1989) with slight modifications by using DNAzol reagent (Invitrogen, USA), instead of sodium dodecyl sulphate (SDS) and proteinase K. The ZFX and SRY genes were selected respectively as X- and Y-chromosome specific genes and PCR was carried out with primers described by Tan et al., (2015).

Conventional PCR was carried out with an optimized 10 µl reaction containing 5 µl of 2 x PCR master mix (Amplicon), 10 picomoles/ µl of 1 µl each of forward and reverse primer, 50 ng/ µl of 1 µl template DNA and 2 µl of nuclease free water (NFW). PCR conditions were 5 min of initial denaturation at 95°C, followed by 35 cycles of 94°C for 30 sec (denaturation), 62°C for 30 sec (annealing) and 72°C for 30 sec (extension) and 10 min of final extension at 72°C. PCR amplificons were verified by using horizontal submarine gel electrophoresis with 2 per cent w/v agarose, visualized under UV illuminator (Bio-Rad Laboratories Inc., USA) and images were stored. Specific gene amplification was identified by comparing the size of the product with 100 bp DNA ladder marker. 

In addition, real-time PCR was carried out in Bio-Rad CFX96 system with amplification profile of initial denaturation at 95°C for 1 min, denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, extension at 72°C for 30 sec and melt curve at 72-98°C every 0.2°C/sec. The reaction mixture (10 µl) contained 5 µl of SYBR green master mix (TB Green), 1 µl (10 picomoles) of forward primer, 1 µl (10 picomoles) of reverse primer, 1 µl (50 ng) of sample DNA and reaction volume made up to 10 µl with nuclease free water (NFW). Standard curves for ZFX and SRY genes were generated using purified PCR products ranging from 1 x 106 to 1 x 102 copy numbers. Copy numbers of each gene calculated separately and converted into percentages by using the formula given by Parati et al., (2006).

Chromosomal analysis of female and male calves, born co-twin, to an indigenous cow sired by a Jersey crossbred revealed leukocyte chimerism of 60, XX / 60, XY on the basis of giemsa staining and morphology of the sex chromosomes (Fig 2). Out of 200 spreads screened, 107 (53.45 per cent) spreads were showing 60, XX cell line, while the remaining (46.55 per cent) spreads were showing 60, XY cell line in the female calf. On the other hand, 109 (54.5 per cent) spreads were found to possess 60, XX cell line and 91 (45.5 per cent) with 60, XY cell line, out of 200 spreads analyzed in the male calf (Table 1).





Though, cytogenetic analysis is the traditional method, it is still effectively used to diagnose the freemartin condition. Diagnosis by cytogenetic testing was proved to be 95-99 per cent accurate when 100 metaphase spreads were studied (Dunn and Johnson,1972; McNiel et al., 2006). The present case was also confirmed through counting 200 metaphase spreads and was further confirmed by molecular diagnostic tools by screening for X- and Y-specific genes and comparing the results with healthy animals.

Molecular analysis through PCR amplification of ZFX and SRY genes revealed the presence of both the genes in female and male calves upon 2 per cent agarose gel electrophoresis (Fig 3). In further, real-time PCR analysis carried out with SYBR green master mix resulted in identification of absolute proportion of ZFX and SRY genes presented in both the co-twins (Fig 4). The expression levels (in percentage) of ZFX and SRY genes in freemartin and healthy calves are given in Table 1. In the freemartin calves, the expression of ZFX gene was found to be higher (i.e., 81.66 and 88.35 per cent in female and male co-twins respectively). While, the per cent expression of ZFX gene was 100 per cent in a normal female and 50.75 per cent in a healthy male calf.        



         

The conventional PCR results revealing the presence of both ZFX and SRY genes in male and female co-twins corroborated with the earlier reports (Pourjafar et al., 2012; Demyda-Peyras et al., 2014; Kozubska-Sobocinska et al., 2019), as evident from the sizes of the PCR products. The identification of SRY gene with real-time PCR gave even more accurate results as observed by Qiu et al., (2018) and Kozubska-Sobocinska et al., (2019). In the present study, the relative content of ZFX and SRY genes in female (81.66 and 18.34% respectively) and male co-twin (88.35 and 11.65% respectively) revealed the high expression levels of ZFX gene compared to normal male and female calves. Unlike conventional PCR, SYBR green assay gave the definitive and differential gene expression levels of ZFX and SRY genes, which additionally supported the leukocyte chimerism of freemartin and male co-twin identified through cytogenetic screening. Dunn et al., (1979) and Padula (2005) reported that presence of minimal defetcts with slightly reduced fertility in male co-twins compared to female co-twin heifer.

Freemartin condition incurs an indirect loss to the dairy industry and it is a not an hindrance to genetic progress. Diagnosis of freemartin calves and male co-twins is necessary immediately after birth to save the farmer’s time and amount spent towards the maintenance of these unproductive animals. The diagnosis of the condition based on clinical signs such as tuft of hair on ventral commissure, short length of vagina, abnormally developed clitoris or external genetalia is not possible, as many of the signs may not manifest immediately after birth. In present study, the male co-twin showed 60, XX/60, XY chimerism, but assessment of fertility could not be done at the early age. Numerous studies had demonstrated that leukocyte chimerism in males associated with lower fertility parameters, reduced non-return rates, lower sperm concentration and motility and also higher degenerative alterations in testicular structures (Cribiu and Popescu, 1982; Switonski et al., 1991). The reason attributed to the biopotential nature of primordial-gonadal cells of fetus, that it could differentiate into either male or female (Ottolenghi et al., 2007; Norling et al., 2013; Piprek et al., 2017). The presence of SRY gene initiates testicular differentiation via up-regulation of SOX9 gene. As well as lower levels of SRY in it alics gene expression causes inadeqaute SOX9 in it alics gene levels, which would be deficit to initiate testicular differentiation (Rebourcet et al., 2014; Ryan, 2014). Once the expression of SOX9 gene reaches to optimum level, the biopotential-gonadal cells differentiate into leydig and sertoli cells (Shoemaker et al., 2007; Benko et al., 2011). However, in the present case, both the calves showed low SRY gene expression compared to normal animals and even then, one fetus had become differentiated into a female and other as male. This may be due to formation of anastamosis between the fetuses after the period of initial differentiation of reproductive organ (Szczerbal et al., 2014).

The cytogenetic analysis, besides, being successfully utilized to diagnose the freemartin condition, the Real-Time qPCR gave a confirmative diagnosis through distinguishing the functionality of ZFX and SRY genes.

The financial support provided by Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai is gratefully acknowledged.

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