Different response to epidermal growth factor of hepatocytes in cultures isolated from male or female rat liver. Inhibitor effect of estrogen on binding and mitogenic effect of epidermal growth factor

Abstract

Deoxyribonucleic acid (DNA) synthesis in hepatocytes isolated from the livers of male and female rats has been compared in monolayer culture. Plating efficiency, DNA and protein content, viability, and morphologic appearance were the same in cultures prepared with hepatocytes isolated from male or female rats. Epidermal growth factor (EGF)-induced DNA synthesis was significantly higher in hepatocytes from male rats than in hepatocytes from female rats. This was the case whether hepatocytes were isolated from normal or partially hepatectomized male or female rats. Hepatocytes isolated from regenerating liver synthesize more DNA than those isolated from normal liver in response to EGF. This increased response to EGF in hepatocytes derived from regenerating liver was relatively the same for male- and female-derived hepatocytes, but the magnitude of the response was considerably higher in male-derived hepatocytes. In contrast, in vivo DNA synthesis in the liver remnant after partial hepatectomy was similar in male and female rats if measured 24 h after the operation. A comparison of EGF binding to male- and female-derived hepatocytes maintained in primary culture indicated a lower number of high-affinity receptors for EGF in the female hepatocytes. The addition of estrogen to primary cultures of hepatocytes isolated from male rats inhibited EGF binding as well as EGF-induced DNA synthesis. Our studies show significant differences in DNA synthesis in response to EGF when male and female hepatocytes are compared in primary culture. The regenerative response after partial hepatectomy, on the other hand, was the same in male and female rats. Thus, our studies indicate that the sex of the donor, rat is important when hepatocytes in culture are used for a variety of studies, such as hepatocyte metabolism, induction and control of DNA synthesis, and hepatocarcinogenesis. In addition, our results indicate that caution is advised when inferences are made from in vitro findings for in vivo conditions.

Figure 1

Labeling index of hepatocytes from male and female rats. Hepotocyte isolation has been described in Materials and Methods. Cells were plated in MEM supplemented with 5% FCS and insulin. After a 3-h attachment period, the medium was changed to serum-free MEM and hormones were added as indicated. Exposure to [³H]thymidine was from 48 to 72 h. Individual culture dishes were processed for autoradiography. In each dish a total of 1000 cells were counted to obtain the percentage of labeled cells. Each bar represents the average of 5 dishes ± SD. Open bars, MEM: hatched bars, MEM + EGF: solid bars, MEM + insulin + EGF.

Figure 2

Examples of labeled hepatocytes exposed to [³H]thymidine for 24 h from male rats grown in MEM supplemented with insulin and EGF for 72 h and stained for glucose-6-phosphatase. The nuclei are filled with black grains of [³H]thymidine. The dark color of the cytoplasm indicated the positive stain. Phase contrast, ×200.

Figure 3

The effect of different EGF levels on DNA synthesis in hepatocytes from male or female rats. Conditions for the assay were the same as for Table 1. The medium was MEM with a constant level (10⁻⁷ M) of insulin. Exposure to [³H]thymidine was from 24 to 48 h or from 48 to 72 h. Values shown were from six different determinations and two different hepatocyte preparations with the SD indicated by the t-bars. ○-○, male hepatocytes at 48 h; ●-●, male hepatocytes at 72 h; △-△, female hepatocytes at 48 h; ▲-▲, female hepatocytes at 72 h.

Figure 4

Epidermal growth factor binding. Scatchard analysis obtained from male and female hepatocytes. Culture and binding conditions are reported in Materials and Methods. ●-●, male hepatocytes: △-△, female hepatocytes.

Figure 5

Epidermal growth factor binding. Scatchard analysis obtained from male. △-△, and female, ●-●, hepatocytes prepared 24 h after 70% hepatectomy.

Figure 6

Epidermal growth factor binding. Scatchard analysis obtained from male hepatocytes incubated for 1 h without, ●-●, and with, ○-○, 10 pg/ml of estradiol.