X -inactive specific transcript (Xist) and X chromosome inactivation in the bovine species

X-inactive specific transcript (Xist) which is thought to be a major factor in regulating X-chromosome inactivation in female mammals, is known to be expressed in male germ cells during spermatogenesis. Expression of Xist and the status of the X-linked genes including glucose 6-phosphate dehydrogenase (G6pd), hypoxanthine phosphoribosyl transferase (Hprt), and zinc finger protein gene ( Zfx), in gonads and nongonadal tissues of male and female fetuses was compared using the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) to test if the expression of Xist leads to the inactivation of these genes in bovine testes of fetuses between 85 to 95 days of gestation. Results showed that the levels of expression of X-linked genes in fetal testes were comparable to those in somatic cells of female fetuses even though Xist expression in fetal testis was evident at this stage of development. Tests on the expression pattern of five X-linked genes, including the bovine homologue of the "selected mouse cDNA on the X" (Smcx), in cell cultures of chromosomally normal bovine fetuses and X chromosome aneuploids showed that G6pd, Hprt and Smcx are subject to inactivation whereas Zfx escapes inactivation in bovine cells. Furthermore, these studies showed that Xist expression increases in keeping with the number of X chromosomes present in the cells, and that it is expressed in normal male (XY) cell cultures. Based on this unexpected finding that male somatic cells which normally do not undergo X inactivation express Xist RNA, a strand-specific RT-PCR approach was undertaken to determine whether the amplicon observed in male bovine cells is Xist per se or its antisense RNA. The results revealed that the RNA detected in fetal and adult bovine testes and nongonadal tissues and cell cultures of males is exclusively that transcribed by the antisense strand while both sense and antisense RNAs are expressed in bovine female cells. The observation that the transcript detected in fetal and adult bovine testes in this study (and probably in the testes of mouse and man by other investigators in previous studies) represents Xist antisense RNA while it is coexpressed with the sense strand of Xist in female cells, leads us to hypothesize that Xist antisense RNA may have a common role to play in both sexes, and that it may be to preserve one X chromosome in an "uninactivated" state.