Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells

Abstract

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.

Figure 1.

Male and female cells show differences in level of cell death induced by different insults. Cell death measured by trypan blue exclusion. Dark bars, male; light bars, females; 10.5, cells from whole embryos; 17.5, cells from ED17.5 kidney; day 4, kidney cells from PN4 mice; day 17, kidney cells from PN17 mice. A) Amount of cell death in control primary mixed cell cultures (controls) from ED10.5 whole embryos, ED17.5 kidney, and PN4 and PN17 kidney; male and female cells exposed to culture medium (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) only. Cells show no sex differences in basal level of cell death. Inset: native polyacrylamide gel with PCR amplification (see Materials and Methods) of Zfy and Zfx, indicative of X- and Y-chromosome-specific genes. Males (right) possess both bands; females (left) have only a single band. B) Cultured cells as in A exposed to 400 μM ethanol. Higher female cell sensitivity to ethanol is seen in cells from ED10.5 whole-embryo, ED17.5 kidney, and PN17 kidney mixed cell cultures, but not in mixed cells cultures from PN4 kidney. C) Cultured cells as in A exposed to 20 μM CPT show female-biased sensitivity at ED10.5 and PN17, but no differences are seen for ED17.5 and PN4 kidney mixed cell cultures. D) Cultured cells as in A infected with influenza A with a multiplicity of infection of 5 show no sex differences in amount of cell death sex, but older cells are more sensitive. Difference at ED10.5 for influenza A infection was not significant by Student’s t test in triplicate experiments. Data represent percentage total cell death for all conditions.

Figure 2.

Cells from male and female ED10.5 whole embryos behave similarly in culture and die by apoptosis. Cells from experiments illustrated in Fig. 1 were used for this analysis as described in Material and Methods. A, B) Light microscopic images of male (A) and female (B) cells of ED10.5 whole embryo before stress possess similar morphology (shape and size) and rate of growth. C–H) Images are from female cells only, as male cells showed identical patterns under the same conditions (data not shown). C, D) Cells labeled with Hoechst stain for DNA condensation and fragmentation; control cells (C) show no chromatin condensation, indicative of apoptosis, before stress, but condensation (arrows) was seen in both female (D) and male cells exposed to 400 μM ethanol. Similar results were seen when cells were exposed to CPT or influenza virus (not shown). E, F) Cells labeled with anti-active caspase-3 antibody, indicative of apoptotic cell death; no caspase-3 activation is seen under control conditions (E) but caspase-3 is activated (arrows) in ethanol-treated cells (F). G, H) Similarly, cells labeled with TUNEL, indicative of DNA fragmentation. No DNA fragmentation is seen under control conditions (G), while DNA fragmentation (arrows) is present in ethanol treated cells (H). Original magnifications are indicated.

Figure 3.

All cells are derived from ED10.5 whole embryos. Cell death is measured by trypan blue exclusion. Dark bars, male; light bars, females. A) ED10.5 mixed cells were maintained in culture medium for 48, 96, and 144 h. There were no differences between male and female control cells at basal level, although cell death increased with time of culture. B) ED10.5 whole-embryo cell cultures were passed and exposed after passages 2, 4, 6, and 8 to 400 μM ethanol and then averages of 3 experiments were plotted. Female sensitivity bias was maintained beyond the first few passages, and rates of death did not differ significantly from passage to passage. C) Influence of estradiol was analyzed by coexposing cells to the stressor ethanol and estradiol. ED10.5 mixed cell cultures exposed to control conditions (control), 400 μM ethanol (ethanol), 5 nM estradiol (estrogen), or both (ethanol estrogen). Female cells maintained the higher sensitivity to ethanol-induced cell death when compared with males, and estrogen did not affect cell death in either sex; however, estrogen completely protected cells of both sexes against ethanol (ethanol estrogen). D) ED10.5 mixed cell cultures were exposed to control culture conditions (control), 400 μM ethanol (ethanol), 5 nM testosterone (testosterone), or both (ethanol testosterone). Female cells maintained higher levels of death when exposed to ethanol alone. Testosterone alone had no effect on male cultures but was toxic to female cells. Testosterone was slightly protective against ethanol for male cells but not for female cells (ethanol testosterone). All treatments were done in triplicate. *P < 0.05; Student’s t test. Data represent percentage total cell death for all conditions.

Figure 4.

Sex differences in gene expression in unstressed ED10.5 whole embryos. A) Virtual reality views of paired male/female embryo microarray intensity data created using the BioMiner VT module. Each sphere represents intensity data from one channel of paired male/female microarray. Blue spheres represent male data, pink represent female data. VR representation of all clones (AA), 51 differentially expressed clones (AB), and the 50 differentially expressed clones with the clone for X-ist removed (AC). B) qRT-PCR verification of sample genes from the microarray. Source of cDNA was whole male or female embryo homogenate at ED10.5. Samples were normalized at the RNA and cDNA levels for equal cDNA loading. Gray bars, male; white bars, female. Experiments were done in triplicate. Individual CT points are shown together with mean values. *P < 0.05; Student’s t test. Ordinate is average CT of fluorescence detection for real-time PCR. Each increment in CT indicates a starting transcript concentration ∼0.5× that of CT-1. X-ist was expressed significantly more in female embryos, while ERα and Cyp7b1 were higher, and GAPDH was not sexually dimorphic.

Figure 5.

Gene activity is sexually dimorphic in standard cell culture but can be altered by additives. Cells exposed to 400 μM ethanol and or in combination with estradiol as in Fig. 3 were used for analysis of gene expression. Gray bars, male; white bars, female. A) X-ist sex differences were maintained during cell culture for ED10.5 whole-embryo control cultures (control). Ethanol treatment (400 μM) did not alter gene expression, while 5 nM estradiol with or without ethanol suppressed sex differences in the expression of X-ist, as a result of a loss in transcript in female cells and an up-regulation of transcription in male cells. B) Differences in Cyp7b1 expression (female higher) were maintained in basal culture (control). Estradiol with or without ethanol reversed gene expression, whereby male transcription was up-regulated, while female transcription was down-regulated. Ethanol alone eliminated sex dimorphism in Cyp7b1 expression. C) ERα expression was independent of sex regardless of culture conditions used. D) Housekeeping gene GAPDH transcript levels were independent of sex under control culture conditions (control) and in the presence of both estrogen and ethanol, while individually estradiol and ethanol stimulated significantly more transcript in male cells. Scale in D differs from other panels because of high transcript levels for the gene. *P < 0.05, male vs. female in each condition.