Testosterone-dependent transgene expression in the liver of the CAG-lacZ transgenic rat

Abstract

Many endogenous gene expressions in the liver are well known to be predominant in males, compared with those of females. In contrast, the fate of hepatic transgene expression between sexes is not fully understood. Here we studied whether sex hormones changed hepatic transgene expression in the ubiquitous CAG promoter-driven lacZ transgenic (Tg) rat. Both sexes of CAG-lacZ Tg rats received gonadectomy. Liver biopsy was taken weekly to determine the change of transgene expression. Histological result of adult males showed mosaic lacZ expression but it was negative in adult females, while livers in neonatal stage showed comparable expression of lacZ. Other organs exhibited equal expression in both sexes. At 2 weeks after castration, lacZ expression in male liver was significantly decreased and became negative after 4 weeks while no significant difference was observed in the lacZ expression pattern in other organs. After ovariectomy, lacZ expression in female liver remained undetectable. Moreover, testosterone treatment to gonadectomized rats of both sexes could enhance lacZ expression in the liver. In summary, we report that CAG-lacZ Tg rats demonstrate sexual dimorphism of transgene expression specifically only in the liver. Testosterone administration mediated upregulation of liver lacZ expression. Our findings suggested that androgen, especially testosterone, plays an important role in the hepatic transgene expression.

Figure 1

Transgene expression in the liver of CAG-lacZ Tg rats. (A) β-Gal protein was examined by X-gal staining and confirmed by immunohistochemistry (insets). In female neonates (c), the transgene expression is comparable to that in males (a). In 8-week-old adults, β-gal expression is maintained in male Tg rats (b), whereas it is lost in females (d). Scale bars: 100 μ. (B) Bars represent the mean ± SD of β-gal concentration. Liver lysates from adult non-Tg rats, neonates, and 8-week-old CAG-lacZ Tg rats of both sexes (n = 3 each) were quantitated using an ELISA method. Neonatal expression does not differ significantly between sexes. However, in adulthood, males have significantly higher (22-fold) β-gal expression than females (*p < 0.005).

Figure 2

Histological changes of hepatic transgene expression after gonadectomy detected by X-gal. In male rats (A–C), compared with before gonadectomy (A), the β-gal level decreased significantly by the second week and disappeared by the fourth week (B and C, respectively) after castration. Ovariectomized rats after 2 and 4 weeks (E and F, respectively) remained negative as in the intact females (D). Bars: 100 μm.

Figure 3

Quantitative analysis of transgene expressions in liver after gonadectomy. Liver lysates from four animal groups (n = 3 each): group 1, intact male rats; group 2, castrated rats; group 3, intact female rats; and group 4, ovariectomized rats. The animals were analyzed for β-gal concentration by ELISA before gonadectomy and 2 weeks and 4 weeks postgonadectomy, respectively. In castrated male rats, β-gal was significantly lower after 2 weeks (*p < 0.01) and was much lower after 4 weeks (**p < 0.001). There was no significant change at any time point in the ovariectomized rats and in both nongonadectomized groups (p > 0.05).

Figure 4

Sex hormones influence hepatic transgene expression in CAG-lacZ Tg rats. In male rats (4A, a–c), injection of testosterone (T) to the castrated animals upregulated β-gal expression (b), compare to the negative expression in the control castrated rats (a). Administration of 17β-estradiol (E₂) to intact male rats downregulated the transgene expression (c). In female rats (4A, d–f), administration of testosterone displayed upregulation of β-gal expression, compared to the negative expression in the controlled intact females (d). The ovariectomized rats (e) show higher transgene expression than the intact rats (f) after testosterone injection. Bars: 100 μm. (B) Bar graph represents numbers of β-gal-positive hepatocyts indicated as mean ± SD. Male liver showed increase β-gal-positive cells after reintroduction of testosterone into castrated animals (†p < 0.001), and high-dose 17β-estradiol injection led to the decrease in β-gal-positive hepatocytes, compared to male control (**p < 0.001). (C) Bars represent that the β-gal-positive hepatocytes in intact female rats after testosterone injection was significantly lower than those from ovariectomy plus testosterone group (*p < 0.05).

Figure 5

Comparison of lacZ and intrinsic liver gene expressions before and after gonadectomy. lacZ transgene, CYP4A2, and CYP3A18 genes from liver cytochrome P450 (CYPs) family, which are expressed in male-predominant manner, were evaluated semiquantitatively by RT-PCR. The extracted total mRNAs from liver tissues were amplified and PCR products of the above genes and GAPDH (internal control) were shown in an ethidium bromide-stained agarose gel. After castration, mRNAs from lacZ and CYP3A18 were markedly decreased whereas CYP4A2 mRNAs in pre- and postcastrated lanes were comparable. Lane M, adult male rat; lane Cx, 4-week-castrated male; lane F, adult female; lane Ovx, 4-week-ovariectomized female; lane Neg, negative control.