BACKGOUND:The male-specific region of chromosome-Y (MSY) contributes to phenotypes outside of testis development and has a high rate of evolution between mammalian species. With a lack of genomic crossover, MSY is one of the few genomic areas under similar variation and evolutionary selection in inbred and outbred animal populations, allowing for an assessment of evolutionary mechanisms to translate between the populations.METHODS:Using next-generation sequencing, MSY consomic strains, molecular characterization, and large-scale phenotyping, we present here regions of MSY that contribute to inbred strain phenotypes.RESULTS:We have shown that (1) MSY of rat has nine autosomal gene transposition events with strain-specific selection; (2) sequence variants in MSY occur with a 1.98-fold higher number of variants than other chromosomes in seven sequenced rat strains; (3) Sry, the most studied MSY gene, has undergone extensive gene duplications, driving ubiquitous expression not seen in human or mouse; (4) the expression profile of Sry in the rat is driven by the insertion of the Sry2 copy into an intron of the ubiquitously expressed Kdm5d gene in antisense orientation, but due to several loss of function mutations in the Sry2 protein, nuclear localization and transcriptional control are decreased; (5) expression of Sry copies other than Sry2 in the rat overlaps with the expression profile for human SRY; (6) gene duplications and sequence variants (P76T) of Sry can be selected for phenotypes such as high blood pressure and androgen receptor signaling within inbred mating; and most importantly, (7) per chromosome size, MSY contributes to higher strain-specific phenotypic variation relative to all other chromosomes, with 53 phenotypes showing both a male to female and consomic cross significance.CONCLUSION:The data presented supports a high probability of MSY genetic variation altering a broad range of inbred rat phenotypes.

The population genetic consequences of nearest- neighbor pollination in an outcrossing plant species were investi- gated through computer simulations. The genetic system consisted of two alleles at a single locus in a self-incompatible plant that mates by random pollen transfer from a neighboring individual. Beginning with a random distribution of genotypes, restricted pol- len and seed dispersal were applied each generation to 10,000 in- dividuals spaced uniformly on a square grid. This restricted gene flow caused inbreeding, a rapid increase in homozygosity, and striking microgeographic differentiation of the populations. Patches of homozygotes bordered by heterozygotes formed quickly and persisted for many generations. Thus, high levels of inbreeding, homozygosity, and patchiness in the spatial distribution of geno- types are expected in plant populations with breeding systems based on nearest-neighbor pollination, and such observations re- quire no explanation by natural selection or other deterministic forces.

Background Gene copy number variation plays a large role in the evolution of genomes. In Rattus norvegicus and other rodent species, the Y-chromosome has accumulated multiple copies of Sry loci. These copy number variations have been previously linked with changes in phenotype of animal models such as the spontaneously hypertensive rat (SHR). This study characterizes the Y-chromosome in the Sry region of Rattus norvegicus , while addressing functional variations seen in the Sry protein products. Results Eleven Sry loci have been identified in the SHR with one ( nonHMG Sry ) containing a frame shift mutation. The nonHMGSry is found and conserved in the related WKY and SD rat strains. Three new, previously unidentified, Sry loci were identified in this study ( Sry3BII , Sry4 and Sry4A ) in both SHR and WKY. Repetitive element analysis revealed numerous LINE-L1 elements at regions where conservation is lost among the Sry copies. In addition we have identified a retrotransposed copy of Med14 originating from spliced mRNA, two autosomal genes ( Ccdc110 and HMGB1 ) and a normal mammalian Y-chromosome gene ( Zfy ) in the Sry region of the rat Y-chromosome. Translation of the sequences of each Sry gene reveals eight proteins with amino acid differences leading to changes in nuclear localization and promoter activation of a Sry-responsive gene. Sry-β (coded by the Sry2 locus) has an increased cytoplasmic fraction due to alterations at amino acid 21. Sry-γ has altered gene regulation of the Sry1 promoter due to changes at amino acid 76. Conclusions The duplication of Sry on the Rattus norvegicus Y-chromosome has led to proteins with altered functional ability that may have been selected for functions in addition to testis determination. Additionally, several other genes not normally found on the Y-chromosome have duplicated new copies into the region around the Sry genes. These suggest a role of active transposable elements in the evolution of the mammalian Y-chromosome in species such as Rattus norvegicus.

The testis determining protein, Sry, has functions outside of testis determination. Multiple Sry loci are found on the Y-chromosome. Proteins from these loci have differential activity on promoters of renin-angiotensin system genes, possibly contributing to elevation of blood pressure. Variation at amino acid 76 accounts for the majority of differential effects by rat proteins Sry1 and Sry3. Human SRY regulated rat promoters in the same manner as rat Sry, elevating Agt, Ren, and Ace promoter activity while downregulating Ace 2. Human SRY significantly regulated human promoters of AGT, REN, ACE2, AT2, and MAS compared to control levels, elevating AGT and REN promoter activity while decreasing ACE2, AT2, and MAS. While the effect of human SRY on individual genes is often modest, we show that many different genes participating in the renin-angiotensin system can be affected by SRY, apparently in coordinated fashion, to produce more Ang II and less Ang-(1–7).

The SHR Y chromosome has loci which are involved with behavioral, endocrine and brain phenotypes and respond to acute stress to a different degree than that of the WKY Y chromosome. The objectives were to determine if WKY males with an SHR Y chromosome (SHR/y) when compared to males with a WKY Y chromosome would have: 1. a greater increase in systolic and diastolic blood pressures (BP), heart rate (HR), and locomotor activity when placed in an open field environment and during an acute stress procedure; 2. enhanced stress hormone responses; 3. greater voluntary running; and 4. increased brain Sry expression. The SHR/y strain showed a significant rise in BP (32%) and HR (10%) during the open field test and exhibited higher BP (46% change) during air jet stress. SHR/y had higher locomotor activity and less immobility and had increased stress induced plasma norepinephrine and adrenocorticotrophic hormone and 3–4× more voluntary running compared to WKY. Differential Sry expression between WKY and SHR/y in amygdala and hippocampus was altered at rest and during acute stress more than that of WKY. Evidence suggests that this animal model allows novel functions of Y chromosome loci to be revealed. In conclusion, a transcription factor on the SHR Y chromosome, Sry, may be responsible for the cardiovascular, endocrine and behavioral phenotype differences between SHR/y and WKY males.

The Sry locus on the mammalian Y chromosome is the developmental switch responsible for testis determination. Inconsistent with this important function, the Sry locus is transcribed in adult males at times and in tissues not involved with testis determination. Sry is expressed in multiple tissues of the peripheral and central nervous system. Sry is derived from Sox3 and is similar to other SOXB family loci. The SOXB loci are responsible for nervous system development. Sry has been demonstrated to modulate the catecholamine pathway, so it should have functional consequences in the central and peripheral nervous system. The nervous system expression and potential function are consistent with Sry as a SOXB family member. In mammals, Sox3 is X-linked and undergoes dosage compensation in females. The expression of Sry in adult males allows for a type of sexual differentiation independent of circulating gonadal hormones. A quantitative difference in Sox3 plus Sry expression in males vs. females could drive changes in the transcriptome of these cells, differentiating male and female cells. Sry expression and its transcriptional effects should be considered when investigating sexual dimorphic phenotypes.

BACKGROUND:Sex-determining region Y (Sry) is a transcription factor. Our research group has shown that there are multiple copies of Sry in Wistar-Kyoto (WKY) and spontaneous hypertensive (SHR) rats, and that they have novel functions separate from testes determination. OBJECTIVE:We hypothesized that exogenously delivered Sry3 to the normotensive WKY male kidney would activate the renin-angiotensin system (RAS) and raise blood pressure (BP), based on previous in vitro studies. METHODS:Sry3 or control vector was electroporated to the left kidney of male WKY rats and the following measurements were taken: BP by telemetry, renin-angiotensin measures by radioimmunoassay, plasma and tissue catecholamines by HPLC with electrochemical detection, sodium by flame photometry, and inulin by ELISA. RESULTS:Sry3 increased BP 10 to 20 mm Hg compared with controls (P < 0.01) and produced a significant 40% decrease in urine sodium compared with controls (P < 0.05). Sry3 increased renal angiotensin II and plasma renin activity by >100% compared with controls (P < 0.01 and P < 0.05, respectively). CONCLUSION:The findings presented here confirm and extend the argument for Sry3 as one of the genes responsible for the SHR hypertensive Y chromosome phenotype and are consistent with increased tissue RAS activity due to Sry3 and increased sodium reabsorption.

The Sry locus on the mammalian Y chromosome is the developmental switch responsible for testis determination. Inconsistent with this important function, the Sry locus is transcribed in adult males at times and in tissues not involved with testis determination. Sry is expressed in multiple tissues of the peripheral and central nervous system. Sry is derived from Sox3 and is similar to other SOXB family loci. The SOXB loci are responsible for nervous system development. Sry has been demonstrated to modulate the catecholamine pathway, so it should have functional consequences in the central and peripheral nervous system. The nervous system expression and potential function are consistent with Sry as a SOXB family member. In mammals, Sox3 is X-linked and undergoes dosage compensation in females. The expression of Sry in adult males allows for a type of sexual differentiation independent of circulating gonadal hormones. A quantitative difference in Sox3 plus Sry expression in males vs. females could drive changes in the transcriptome of these cells, differentiating male and female cells. Sry expression and its transcriptional effects should be considered when investigating sexual dimorphic phenotypes.

Background and objective We demonstrated that the Sry gene complex on the spontaneously hypertensive rat (SHR) Y chromosome is a candidate locus for hypertension that accounts for the SHR Y chromosome blood pressure effect. All rat strains examined to date share six Sry loci, and a seventh Sry locus (Sry3) appears to be unique to SHR male rats. Previously, we showed that Sry1 increased activity of the tyrosine hydroxylase promoter in transfected PC12 cells, and Sry1 delivered to adrenal gland of Wistar–Kyoto (WKY) rats increased blood pressure and sympathetic nervous system activity. The objective of this study was to determine whether renin–angiotensin system genes participate in Sry-mediated effects. Method Sry expression vectors were co-transfected into CHO cells with luciferase reporter constructs containing promoters of angiotensinogen (Agt −1430/+22), renin (Ren −1050/−1), angiotensin-converting enzyme (ACE) (ACE −1677/+21) and ACE2 (ACE2 −1091/+83). Results Sry1, Sry2 and Sry3 differentially upregulated activity of the promoters of angiotensinogen, renin and ACE genes and downregulated ACE2 promoter activity. The largest effect was seen with Sry3, which increased activity of angiotensinogen promoter by 1.7-fold, renin promoter by 1.3-fold, ACE promoter by 2.6-fold and decreased activity of ACE2 promoter by 0.5-fold. The effect of Sry1 on promoter activity was significantly less than that of Sry3. Sry2 activated promoters at a significantly lower level than Sry1 did. The result of either an additive effect of Sry regulation of multiple genes in the renin–angiotensin system or alterations in expression of a single gene could favor increased levels of Ang II and decreased levels of Ang-(1-7). Conclusion These actions of Sry could result in increased blood pressure in males and contribute to sex differences in blood pressure.

The Sry gene has a well established role in testes determination. However, we have reported Sry gene expression in the adult adrenal gland and that blood pressure, plasma norepinephrine and tyrosine hydroxylase content increased following exogenous delivery of the Sry gene into the adrenal medulla. We have observed changes in Sry expression in stress responsive areas of the brain, and have co‐localized Sry and tyrosine hydroxylase (Th) in both the adrenal medulla and various brain areas. The objective of this experiment was to evaluate the involvement of the androgen receptor on Sry and Th expression in sympathetic ganglia of the rat. We measured gene expression in celiac ganglia of males lacking functional androgen receptors (testicular feminized males, Tfm) and sibling males having normal receptors. Male WKY rats were crossed to King‐Holtzman Tfm carrier females. Litters yielded normal males and Tfm siblings. All animals were terminated at 12 weeks of age and gene expression was measured by quantitative RT‐PCR. Expression of Sry and Th genes in both groups was measured. In all samples expression of Sry paralleled expression levels of Th which suggests that Th expression is regulated by Sry and not through an androgen receptor‐mediated mechanism. (Supported by NIH 1RO1HL71579‐01A3)

Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in SHR and WKY rats with the SHR Y chromosome (SHR/y rat). A candidate gene is Sry, a transcription factor responsible for testes determination which is also expressed in the kidney. We have found multiple functional Sry loci in our WKY and SHR rats. Sry1 and Sry3 exogenously delivered to the kidney raise BP. Sry1 activates SNS indices whereas Sry3 does not. The objective of this study was to determine if exogenous Sry1 or Sry3 delivered to WKY kidney would elevate indices of the renin‐ angiotensin system (RAS). The expression construct Sry1pEF1 (n=8) or Sry3/pEF1 (n=8), or control vector (n=8) was electroporated to the medulla of the left kidney of WKY rats. After 14 and 21 days the kidneys from half of the groups were analyzed for ACE, angiotensin II, renin and plasma renin activity (PRA) was measured. Renal AngII increased by more than 100% after 21 days in the Sry3 kidneys (p<.01) compared to controls. PRA was increased more than 100% after 14 days in the Sry3 kidneys (p<.05) compared to controls but there was no significant difference in renal renin between groups. Renal ACE content decreased after 21 days in the Sry3 kidneys compared to controls (p<.05). These results are consistent with a role for Sry3 in BP elevation due to elevated RAS, whereas Sry1 has more of a BP influence through SNS activity, as we showed earlier ( RO1HL71579‐01A3).

Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in the SHR rat and in WKY rats with the SHR Y chromosome (SHR/y rat). A candidate for this Y chromosome hypertension locus is Sry, a gene that encodes a transcription factor responsible for testes determination. The SHR Y chromosome has six divergent Sry loci. The following study examined if exogenous Sry1 or Sry2 delivered to the kidney would elevate renal tyrosine hydroxylase, renal catecholamines, plasma catecholamines and telemetered BP over a 28 day period. We delivered 50 μg of either the expression construct Sry1/pcDNA 3.1, Sry2/pcDNA 3.1, or control vector into the medulla of the left kidney of normotensive WKY rats by electroporation. Weekly air stress was performed to determine BP responsiveness. Separate groups of animals were tested for renal function and plasma hormone patterns and pharmacological intervention using alpha adrenergic receptor blockade. Pre-surgery baseline and weekly blood samples were taken from Sry1 electroporated and control vector males for plasma renin, aldosterone, and corticosterone. BP was measured by telemetry and tyrosine hydroxylase and catecholamines by HPLC with electrochemical detection.

The Y chromosome of the spontaneously hypertensive rat (SHR) contains a genetic component that raises blood pressure compared with the Wistar-Kyoto (WKY) Y chromosome. This research tests the Sry gene complex as the hypertensive component of the SHR Y chromosome. The Sry loci were sequenced in 1 strain with a hypertensive Y chromosome (SHR/Akr) and 2 strains with a normotensive Y chromosome (SHR/Crl and WKY/Akr). Both SHR strains have 7 Sry loci, whereas the WKY strain has 6. The 6 loci in common between SHR and WKY strains were identical in the sequence compared (coding region, 392-bp 5' prime flanking, 1200-bp 3' flanking). Both SHR strains have a locus (Sry3) not found in WKY rats, but this locus is different between SHR/Akr and SHR/Crl rats. Six mutations have accumulated in Sry3 between the SHR strains, whereas the other 6 Sry loci are identical. This pattern of an SHR-specific locus and mutation in this locus in SHR/Crl coinciding with the loss of Y chromosome hypertension is an expected pattern if Sry3 is the Y chromosome-hypertensive component. The SHR/y strain showed a significant increase in total Sry expression in the kidney between 4 and 15 weeks of age. There are significant differences in Sry expression between adrenal glands and the kidney (15 to 30 times higher in kidneys) but no significant differences between strains. These results, along with previous studies demonstrating an interaction of Sry with the tyrosine hydroxylase promoter and increased blood pressure with exogenous Sry expression, suggest the Sry loci as the hypertensive component of the SHR Y chromosome.

Sry is a transcription factor with a conserved HMG domain that facilitates DNA interactions and nuclear localization, through amino (n) and carboxy (c) terminal nuclear localization sequences (NLS). There are seven Sry loci on the SHR/Akr Y chromosome. Amino acid (aa) differences among Sry proteins are in the nNLS at aa 21 (His vs. Arg), cNLS at aa 76 (Pro vs. Thr), and in the presence/absence of 13 aa in a c‐ terminal activation motif. The objective of this study was to determine if these aa differences between proteins alter nuclear localization. Native, chimeric, mutated and truncated Sry sequences were transfected into CHO cells followed by immunocytochemistry and Western blot analysis of nuclear and cytoplasmic CHO cell fractions. Results showed that Sry2 exhibits both cytoplasmic and nuclear accumulation, while Sry1 and 3 localize only to the nucleus. Sry1 and 3 sequences containing the truncated or deleted activation region exhibit no reduction in nuclear localization, while mutations to the NLS result in cytoplasmic accumulation. Changing His to Arg at aa 21 of Sry1 or Sry3 results in nuclear localization patterns equivalent to native Sry2. Results demonstrate that aa variability in Sry proteins leads to different intracellular localization in vivo and may account for the different phenotypes observed in rats after delivery of exogenous Sry1, Sry2 or Sry3.Support from NIH HL71579 and the University of Akron.

Background The Y-chromosome (Yc) and testosterone (T) increase blood pressure and may also influence renal electrolyte excretion. Therefore, the goal of this study was to determine if the Yc combined with T manipulation could influence renal Na and K excretion. Methods To investigate the role of the Yc and T, consomic borderline hypertensive (SHR/y) and normotensive Wistar-Kyoto (WKY) rat strains were used (15 weeks) in three T treatment groups: castrate, castrate with T implant and gonadally intact males. Urine was collected (24 hrs at 15 weeks of age) for Na and K measurements by flame photometry. RT-PCR was used to demonstrate the presence of renal androgen receptor (AR) transcripts. Plasma T and aldosterone were measured by RIA. In another experiment the androgen receptor was blocked using flutamide in the diet. Results Na and K excretion were decreased by T in SHR/y and WKY. AR transcripts were identified in SHR/y and WKY kidneys. Plasma aldosterone was decreased in the presence of T. Blockade of the AR resulted in a significant increase in Na excretion but not in K excretion in both SHR/y and WKY males. Conclusion T influences electrolyte excretion through an androgen receptor dependent mechanism. There was not a differential Yc involvement in electrolyte excretion between WKY and SHR/y males.