the title of this article, which is attributed to Woody Allen, suggests that sex is a universal truth, but in the experience of the authors of this Editorial Focus, this is rarely the case in scientific experiments, including in physiology, and especially cell physiology. Most investigators have had to reconcile disparate results between experiments conducted under identical conditions and attribute such problems to factors that include the “gods” of science, the season, the weather, or even the wearing of lucky socks. Unless it is the focus of the research, one aspect that is rarely if ever considered is the sex of the cells being used. Do fibroblasts (or other cells) bearing an XY genotype behave the same as cells that are XX? We tend to assume so, but we don't really know the answer. For example, do T-84 cells, derived from a male colon cancer patient, behave the same as Ht-29 colon cancer cells, derived from a female? If experiments with the two different lines give the same answer to a question under study, we are happy with the outcome, but if different results are obtained, studies on one or the other cell lines are usually suspended or abandoned, in favor of the cells where the experiments yielded the expected or exciting results. Although sex hormones target non-reproductive cells and tissues, and most of us know that these hormones are likely present in fetal bovine serum or other serum additives included in media, few of us use charcoal-stripped serum to eliminate the hormones.
However, since 2012, authors of manuscripts published in American Physiological Society (APS) journals have been asked to include the sex of the animals used in their research and to report the sexual identity of cells or other biological materials used in their studies. This requirement has been highlighted in three recent articles published in AJP-Cell and Nature. The first, by Virginia Miller (4), provides useful definitions of sex and gender, pointing out that sex refers to chromosomal identity, whereas gender is a cultural/behavioral construct. For authors submitting manuscripts to AJP-Cell, it is virtually always the former that is the most relevant. Surveys of articles published in scientific journals have revealed that only a minority (around 25%) identify the sex of the cell lines on which the studies were conducted (7). The second article, by Pollitzer (5), highlights the third Gender Summit sponsored by the National Science Foundation that took place in in November 2013 in Washington DC. The focus of this meeting was the effects of sex differences on basic cell regulation, stem cell function, and clinical and pharmaceutical science. The third article, by Shah et al. (6), in the current issue of AJP-Cell, explores the subject of sex from the point of view of chromosomal differences, the role of sexually dimorphic genes (i.e., genes that express differently in males and females independent of the influence of sex hormones), and the impact that these differences can have on the biology of non-gonadal or non-sex-specific cells and tissues.
While it might be assumed that identification of the sex of a cell line would be a simple matter of identifying the XX or XY genotype, it turns out that many supposedly male cell lines have lost the Y chromosome, including the widely used male-derived T-84 colonic carcinoma epithelial cell line. Experiments comparing the biological properties of T-84 and the female Ht-29 cell line reveal few differences; is this because the cells are functionally equivalent, or does this result from the loss of the Y chromosome by the T-84 cells?
Interestingly (especially for one of the authors of this Editorial Focus), the Y chromosome encodes only about 50–60 genes, whereas the X chromosome is estimated to encode 800–900 genes (http://ghr.nlm.nih.gov/chromosome/). These include genes for channels (CLCN5), transporters (ATP7A, SLC9A6), and G protein-coupled receptors (GPR143), among many others. Thus, the sex chromosome genes serve functions other than the simple determination of sex. Further evidence for this is the presence of multiple X-linked conditions, including red-green color blindness, Duchenne muscular dystrophy, and hemophilia.
Sexual dimorphism of gene expression is increasingly appreciated as an underlying factor that contributes to differences in behavior of many somatic cells. Dewing et al. (3) identified 51 genes that were differentially expressed in brains of female mice as compared to male mice, prior to the expression of sex hormones. These included genes for sulfate transport, uridine synthetase, and the IL-7 receptor, among others; few of these genes are expressed on the X chromosome (3). Sexual dimorphism has been particularly well studied in the brain. Dopamine-expressing neurons exhibit different morphology depending on whether they are isolated from male or female rat fetuses and independent of the presence of gonadal hormones (2). Furthermore, cultures of female diencephalic neurons harvested prior to detectable differences in sex hormone levels have higher tyrosine hydroxylase levels than do their male counterparts (1). Microarray data support these observations and show that many thousands of transcripts representing multiple genes have dimorphic expression; as noted by Shah et al., sexual dimorphism may be as high as 70% in some tissues. If the prevalence of dimorphism is so common, then does cell sex matter at all? As Shah et al. emphasize, physiological differences in the functioning of stem cells and the relative resistance of female-derived cells (compared to male-derived cells) to oxidative stress suggest that it does.
The recent and growing emphasis on personalized medicine implies that these differences should be taken into account. Those developing drugs (be they small molecules or biologics) will need to consider that sex differences may underlie differences in responsiveness of different cells used in high throughput screens as well as considering the sex of the patient group to which the drugs are targeted. Sex differences will be particularly important in stem cell-based therapies, such that the sex of both the donor and the recipient should be considered.
How does this issue impact on authors who submit their work to an APS journal? Fortunately, Shah et al. have provided a table of nearly 100 commonly used cell lines with the sex and other basic information included where known. This table should be a helpful reference resource for prospective authors. The reader is also referred to the ATCC website (www.ATCC.org) for additional information about cell lines not included in the article by Shah et al. With all due respect to Woody Allen, sex may be a panacea, but in the context of scientific experiments it can be also be a confounding and enigmatic property of cells.
Preparation of this Editorial Focus was supported in part by National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206 (C. M. Fuller).
No conflicts of interest, financial or otherwise, are declared by the author(s).
C.M.F. and P.A.I. drafted manuscript; C.M.F. and P.A.I. edited and revised manuscript; C.M.F. and P.A.I. approved final version of manuscript.
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