PMC

MSY consomic rats that have been generated. Crosses of male (♂) and female (♀) rat strains have generated six different MSY consomic strains. Abbreviations for rat strains are shown in the first box. Phenotyping of the animal was performed at the designated generation (F11, F2, F9) relative to a male from the strain designated in a circle. For two of the consomic strains, they were crossed with the Tfm rat, containing a loss of function androgen receptor mutation, to assess the segregation of blood pressure contributions due to MSY vs. MSY and hormone signaling

Characterization of the multiple Sry copies in commonly used rat strains. a Schematic breakdown of the domains found in rat SRY showing the N-terminus (red), HMG box (blue), hinge (green), bridge (magenta), and C-terminal (yellow) domains of the protein. Locations of SNVs that allow for the identification of individual copies based on the known SHR sequence are shown below the schematic. b Protein model of the full rat Sry1 protein bound to DNA (gray). Color scheme from a for the various domains are shown on the model. c Detection of the SNVs for the multiple copies of Sry from the genomic sequence reads for multiple male rat strains (SHR, WKY, FHH, FHL, ACI, SR, SS, and F334). The plus sign represents multiple reads with 100 % homology (red boxes), the minus sign represents those with no positive SNVs detected (no color), and nd are copies of Sry that do not contain SNVs allowing for identification using short reads. d–f Real-time PCR of Sry from five tissues for WKY (d), SHR (e), or the SHR/y consomic (f)

SRY blood pressure regulation through androgen receptor and the renin-angiotensin system. a Testosterone-dependent synergistic regulation between SRY and AR is altered by mutations to SRY at the location that separates Sry3 (T) and all other rat and mammalian SRY sequences (P). b Delivery of the Sry3 expression vector (open circle) to the kidney of WKY male rats at day 0 significantly increased blood pressure relative to a control (closed circle) 14 days after vector electroporation. Olmesartan, a RAS inhibitor, administered to control and half of the Sry3-treated animals (closed triangle) at day 14, significantly decreases blood pressure to the same value in both groups. Following removal of olmesartan at day 17, blood pressure increased more rapidly in the Sry3-treated group (closed triangle). Error bars are shown as the SEM of three to four independent animals with asterisk representing a P < 0.05 for blood pressure between the Sry3 and control vector electroporated animals

Expression and function of Sry copies. a Analysis of Sry transcripts (as reads per million sequence reads that contain identifying SNP) from the Rat BodyMap datasets for multiple tissues of males and a control female uterus tissue. Colors correspond to the Sry identifying SNP; Sry1 = blue, Sry2 = red, Sry3’s = green, Sry4’s = cyan, nonHMGSry = magenta. b Analysis of publically available rat RNAseq datasets (excluding the Rat BodyMap datasets) for the SNVs of Sry2 (x-axis) vs. the other non-Sry2 copies (y-axis). Tissues with the highest expression of non-Sry2 copies are labeled in red. c Differences in transcriptional regulation by the SRY2 protein. Alanine mutations to nuclear localization site of Sry1 (Sry1 20-22A) results in a loss of promoter regulation (red). Mutating amino acid 21 from His (found in Sry2) to an Arg (found in Sry1 and Sry3) in the SRY2 protein results in a significant elevation of promoter activity (blue) relative to Sry2. d, e Nuclear localization of Sry constructs shown as Western blot for multiple Sry constructs (d) or quantification of western blot for several constructs (e). For panel d, Lane 1 = Sry1(HMGbox), 2 = Sry1(delPolyQ), 3 = Sry2, 4 = Sry2(−QR), 5 = Sry1, 6 = Sry3, 7 = Sry1(20-22AAA), 8 = Sry1(78-79AA), 9 = Sry1(NoNLS), 10 = Sry1(R21H), 11 = Sry2(H21R), 12 = negative control (empty vector)

High-throughput phenotyping of two consomic panels in the rat. a The percent of significantly altered phenotypes due to crossing each chromosome in two separate consomic panels (BN to FHH in black and BN to SS in red) shown per megabase (Mb) or per ten genes of the chromosome. b Phenotypes identified in a to be significantly altered by the two MSY consomic rats (FHH-YBN/Mcwi in black and SS-YBN/Mcwi in red) were then separated based on if a significant difference between males and females was also seen for one or both strains used to produce the consomic rat. Phenotypes that had a sex difference in one specific strain are listed in each category. Two phenotypes were identified to overlap in the two consomic rats with a BN-specific sex difference, dilator response to acetylcholine EC50 and dilator response to acetylcholine Log EC50. c, d The dilator response to acetylcholine EC50 for male (black) and female (gray) BN and FHH (c) or SS (d) rats showing BN to have the largest sex difference. MSY consomic significantly decreased the response in both FHH (c) and SS (d) consomic rats (red). The chromosome 15 consomic rats (FHH-15BN/Mcwi and SS-15BN/Mcwi) resulted in a greater sex difference for both strains. Error bars represent the SEM of independently tested rats and asterisk represents an adjusted P value <0.05 calculated with Mann-Whitney test followed by a Bonferroni adjustment

Rat MSY genes. a Alignment of the various genes described in this paper onto the RNor 6.0 MSY sequence of Rattus norvegicus. Retroposed genes that align onto the sequence are shown on the top. Ubiquitously expressed genes in the Rat BodyMap dataset are colored in purple while tissue-specific genes are in black. b, c Expression profiling for the F344 Rat BodyMap dataset for Med14Y (b) and Ube2q2Y (c) in multiple tissues of male and female animals. Expression is shown as the number of reads for MSY copy (Med14y/Ube2q2y) divided by X (Med14y) or chromosome 8 (Ube2q2) and Y copies, in such that values of 0.5 are a 50/50 ratio of transcripts from the X/8 and MSYs and a value of 0 is expression from only the X/8-chromosome. Error bars represent the error of four independent RNAseq datasets and asterisk represents a P < 0.05 between male and female rats. d Distribution of SNPs detected in seven male rat strains on the genome. SNP counts on the y-axis of box-and-whisker plot are normalized to the chromosome size in Mb. MSY (red) is shown to have highly elevated number of mutations compared to all other chromosomes