Feminized behavior and brain gene expression in a novel mouse model of Klinefelter Syndrome

Abstract

Klinefelter Syndrome (KS) is the most common sex chromosome aneuploidy in men and is characterized by the presence of an additional X chromosome (XXY). In some Klinefelter males, certain traits may be feminized or shifted from the male-typical pattern towards a more female-typical one. Among them might be partner choice, one of the most sexually dimorphic traits in the animal kingdom. We investigated the extent of feminization in XXY male mice (XXYM) in partner preference and gene expression in the bed nucleus of the stria terminalis/preoptic area and the striatum in mice from the Sex Chromosome Trisomy model. We tested for partner preference using a three-chambered apparatus in which the test mouse was free to choose between stimulus animals of either sex. We found that partner preference in XXYM was feminized. These differences were likely due to interactions of the additional X chromosome with the Y. We also discovered genes that differed in expression in XXYM versus XYM. Some of these genes are feminized in their expression pattern. Lastly, we also identified genes that differed only between XXYM versus XYM and not XXM versus XYM. Genes that are both feminized and unique to XXYM versus XYM represent strong candidates for dissecting the molecular pathways responsible for phenotypes present in KS/XXYM but not XXM. In sum, our results demonstrated that investigating behavioral and molecular feminization in XXY males can provide crucial information about the pathophysiology of KS and may aid our understanding of sex differences in brain and behavior.

Figure 1. The experimental setup used for this study

A. The breeding scheme used to generate SCT mice. Mothers are XXY⁻ and produce two types of eggs: X and XY⁻. Fathers are XY⁻(Sry+) and make four types of sperm: X, X(Sry+), Y^−, and Y⁻(Sry+). The progeny have four possible sex chromosome complements: XX, XY⁻, XXY^−-, or XY⁻Y⁻ all of which can be with or without Sry. B. The three-chambered apparatus for partner preference testing. A stimulus animal (either male or an estrous female) was placed in each lateral chamber. The chambers holding the stimulus animals are separated from the large middle chamber by clear, perforated dividers. The test animal is placed in the middle chamber where it is free to choose to spend time close to either stimulus animal (in the gray incentive zones) or by itself in the middle. Time spent in each incentive zone was recorded and used as a measure of time spent with that stimulus animal. C. Study timeline. The numbers along the scale are the ages (in days) at which the experimental procedures were carried out. At about postnatal day 100, all mice used in the study underwent bilateral gonadectomy and received an implant of a Silastic capsule filled with testosterone. Behavioral testing began a week after surgery. At about 128 days of age and after completion of behavioral testing, blood and tissue collection was performed.

Figure 2. Time spent with the stimulus animal

The boxplot depicts time spent with the stimulus animal of each sex in quartiles. The whiskers represent the maximum and minimum amount of time spent with the stimulus animal in each group. A. XXY, but not XX, males spend significantly less time with the stimulus female compared to XY males. B. Median time spent with the stimulus male is higher in XXY compared to XY males. *p < .05

Figure 3. Feminization of brain gene expression in XXYM

Levels of expression of genes in XXYM were assigned a feminization score based on the similarity to expression levels in XXF relative to XYM, then plotted on a continuum between average XYM and XXF expression in A. the BNST/POA, and C. the striatum. Dots represent the mean score of each gene in XXYM whereas lines represent the standard error of the mean. The color of the dot indicates whether expression of that gene is closer to the male (blue) or female (red) end of the spectrum. Genes considered feminized are indicated by the dashed box. A feminization score of 70 was used as the lower boundary for categorizing a gene as feminized. The expression pattern of feminized genes in B. the BNST/POA and D. the striatum that survive Benjamini-Hochberg correction (FDR=10%) are visualized using a heat map. Genes are clustered based on similarity in their pattern of expression across samples.

Figure 3. Feminization of brain gene expression in XXYM

Levels of expression of genes in XXYM were assigned a feminization score based on the similarity to expression levels in XXF relative to XYM, then plotted on a continuum between average XYM and XXF expression in A. the BNST/POA, and C. the striatum. Dots represent the mean score of each gene in XXYM whereas lines represent the standard error of the mean. The color of the dot indicates whether expression of that gene is closer to the male (blue) or female (red) end of the spectrum. Genes considered feminized are indicated by the dashed box. A feminization score of 70 was used as the lower boundary for categorizing a gene as feminized. The expression pattern of feminized genes in B. the BNST/POA and D. the striatum that survive Benjamini-Hochberg correction (FDR=10%) are visualized using a heat map. Genes are clustered based on similarity in their pattern of expression across samples.

Figure 4. Quantitative RT-PCR confirmation of feminized genes in XXY males

mRNA levels of Eif2s3x and Utx (Kdm6a) in XY males, XX females, and XXY males in both the BNST/POA and the striatum were analyzed. Error bars represent the standard error of the mean from 6-8 biological replicates from each group. Expression is relative to GAPDH and is normalized to XY males. *p < .05 by the Student's t-test.

Figure 5. Determination of genes affected uniquely by being XXY

The Venn diagram shows genes that are differentially expressed (>1.2-fold, p < .05 by Student's t-test) between XXY and XY males (white), and between XX and XY males (black) in A. the BNST/POA and B. the striatum. This procedure identified 20 genes in the BNST/POA and 19 genes in the striatum that may be different in both comparisons because of the common difference in number of X chromosomes (gray).