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 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.

Background: Testosterone (T) and the sympathetic nervous system each contribute to the pathology of hypertension. Altered blood vessel reactivity is also associated with the pathology of high blood pressure. The purpose of this study was to examine the effects of T manipulation in the regulation of resistance-sized blood vessel reactivity.Methods: Adult spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) male rats at 8 weeks of age were used. The rats were divided into groups consisting of gonadally intact controls (CONT), castrate with sham implant (CAST) and castrate with T implant (CAST + T) (n = 6 to 12 per group). Following a short-term period of T treatment (approximately 4 weeks), plasma norepinephrine (NE) and plasma T were assessed by performing high-performance liquid chromatography and RIA, respectively. Resistance-sized mesenteric artery reactivity was assessed on a pressurized arteriograph for myogenic reactivity (MYO), phenylephrine (PE) responsiveness and passive structural mechanics.Results: SHR and WKY males exhibited similar physiological trends in T manipulation, with castration significantly lowering plasma T and NE and T replacement significantly increasing plasma T and NE. T manipulation in general resulted in significant alterations in MYO of second-order mesenteric arteries, with T replacement decreasing MYO in SHR (P < 0.05) compared to CONT, T replacement increasing MYO, and CAST decreasing MYO in WKY rats (P < 0.001) compared to CONT rats. Additionally, PE-induced constriction was significantly altered in both strains following T treatment, with the effective concentration of PE to constrict the vessel to 50% of the total diameter significantly increased in the CAST + T SHR compared to CONT (P < 0.05). Comparisons of passive structural mechanics between SHR and WKY treatment groups indicated in SHR a significantly increased wall-to-lumen ratio and decreased circumferential wall stress compared to WKY treatment groups.Conclusions: These data suggest that T and NE are involved in a complex interaction with both myogenic reactivity and structural alterations of resistance-sized blood vessels and that these factors likely contribute to the development and maintenance of hypertension.

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.

Increased sympathetic nervous system (SNS) activity, testosterone, and spontaneously hypertensive rat Y chromosome (SHR Yc) play a role in a genetic model of hypertension. Male rats with the SHR Yc and Wistar-Kyoto (WKY) autosomes (denoted SHR/y) exhibit these characteristics when compared to rats with the WKY Yc and WKY autosomes (denoted WKY). We hypothesized that chronic social stress will increase blood pressure and SNS activity more in SHR/y males compared to WKY males, resulting in increased myogenic reactivity along with decreased vasoconstriction of small mesenteric arteries. SHR/y and WKY males were housed in strain-specific colonies (10 males with 10 females) or as controls (10 males). Systolic blood pressure (SBP) and blood samples were collected prior to termination. Second-order mesenteric arteries were studied using a pressure arteriograph in which myogenic reactivity and phenylephrine (PE) responsiveness were measured. SHR/y colony SBP, and circulating norepinephrine and testosterone concentrations were elevated compared to control and WKY colony males (p < 0.05). Mesenteric artery myogenic reactivity was increased in SHR/y colony males (p < 0.001). Mesenteric arteries from SHR/y colony males exhibited a significant decrease in PE-induced constriction. Colony social stress elevated both SNS activity and testosterone level which may be responsible for the increased mesenteric artery myogenic reactivity, and SBP as noted in SHR/y males.

Estrogen (E) and testosterone (T) are important in the sexually dimorphic pattern of blood pressure (BP) development. The goal was to examine the effects of endogenous E and exogenous T in the development of hypertension in female spontaneously hypertensive rats (SHR) on a high sodium diet. Female SHR (N = 27, 5-week) were divided into four groups: (1) control (n = 8), (2) ovariectomized (OVX, n = 26), (3) testosterone implants with intact ovaries (T, n = 6), and (4) ovariectomized + testosterone implants (OVX+T, n = 7). T was given immediately after OVX and replaced every two weeks and they were fed a 3% NaCl diet. BP was measured weekly and plasma norepinephrine (NE) analyzed by HPLC. OVX+T females exhibited the greatest elevation in BP (190 ± 4.0 mmHg) compared to controls at 15 weeks of age (140 ± 3.4 mmHg, P < .001) and a pattern of hypertension development similar to that of male SHR. Females with T treatment showed evidence of glomerulosclerosis. In conclusion, T accelerated the development of hypertension similar to the BP pattern observed in males; the presence of ovaries attenuated the T induced increase in BP; T increased renal sodium reabsorption, and T increased glomerulosclerosis.

Background: Testosterone (T) and the androgen receptor (AR) are involved in mechanisms associated with hypertension and vessel reactivity.Objective: To investigate T and the AR on blood vessel reactivity, testicular feminized male (TFM; AR deficient males) and normal androgen receptor (NAR) male rats were used. Therefore, if the functional AR is necessary for plasma T to regulate vessel responsiveness, TFM males will exhibit altered vessel function compared to NAR males.Methods: Adult (16 weeks of age) TFM or NAR males were assigned to the following treatment groups: gonadal intact controls (CONT), castrate (CAST), or castrate with T replacement (CAST+T) with (n=8-10/group).Results: Plasma T followed a consistent pattern with CAST+T elevated compared to CONT and CAST TFM and NAR males. In addition, CAST plasma T was significantly decreased compared to CONT and CAST+T in TFM and NAR males. In a similar manner for systolic blood pressure (SBP), CAST lowered SBP compared to CONT in both NAR and TFM. Following 8 weeks of treatment, second-order mesenteric artery responses to changes in intraluminal pressure (myogenic reactivity) were analyzed using a pressure arteriograph system. Both TFM (P < 0.05) and NAR (P < 0.05) CAST groups revealed a decrease in myogenic reactivity compared to CONT. Following T treatment the TFM CAST+T myogenic reactivity returned to CONT levels, whereas the NAR CAST+T myogenic reactivity increased a further 10%.Conclusion: The results of this study indicate that T differentially regulates mesenteric artery reactivity in TFM and NAR males. Our data also demonstrate that both AR and/or non-AR mediated mechanisms may partially contribute to SBP regulation. (Gend Med. 2011;8:40-52) (C) 2011 Elsevier HS Journals, Inc. All rights reserved.

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.

Background . The vasodilator hydralazine (HYZ) has been used successfully to lower blood pressure (BP) in hypertension. Recently proposed novel mechanisms suggest it may be an important drug in reducing cardiovascular pathology. Methods . The hypothesis was that social stress would increase BP and endocrine factors that lead to increased coronary artery collagen deposition, and that HYZ treatment would reverse these changes. SHR males were compared: controls, colony stress and colony stress with HYZ treatment. Results . BP was significantly elevated in the stress group compared to controls and HYZ reduced the BP compared to controls. Plasma Ang II and T were significantly increased by colony stress compared to controls and HYZ restored both to control values. Collagen deposition in the coronary artery was increased in the colony stress group compared to controls but HYZ treatment restored the collagen to that of control values. Conclusions . HYZ reduced not only BP, but also reduced coronary adventitial collagen. The mechanism of the BP effect is most likely through vasodilation and the collagen reduction may be due to both a direct action of HYZ on collagen synthetic enzymes and indirectly through an effect of testosterone and angiotensin II.

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 oxidative/hydrolytic stability of polyurethanes (PUs) containing exclusively polyisobutylene (PIB), or mixed PIB/polytetramethylene oxide (PTMO), or mixed PIB/polyhexamethylene carbonate (PC) soft segments was investigated. The tensile strengths and elongations of various PUs were determined before and after agitating in 35% HNO3 or 20% H2O2/0.1 M CoCl2 solutions and retentions were quantified. The presence of PIB imparts significant oxidative/hydrolytic resistance. The tensile strength and elongation of PUs containing 70% PIB, or those of mixed PIB/PC soft segments with 50% PIB, remained essentially unchanged upon exposure to HNO3; in contrast, PUs containing mixed PIB/PTMO soft segments with 50% PIB underwent significant degradation. The tensile strength of PUs with mixed PIB/PC (60/10%) soft segment increased after exposure to HNO3, most likely because of oxidative crosslinking of PC segments. PIB/PTMO- and PIB/PC-based PUs and commercially available PUs (Elast-Eon (R) e and Carbothane (R)) were exposed to H2O2/CoCl2 solutions for up to 14 weeks. Although the experimental PIB/PC-based PUs exhibited negligible change in mechanical properties and no surface damage, ElastEon (R) and Carbothane (R) showed significant surface damage. PIB-based polyureas and Bionate (R) were implanted in rats for 4 weeks in vivo, and their biocompatibility was investigated. The biocompatibility of PIB-based materials was superior to Bionate (R). (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2194-2203, 2010

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 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.

Acute and chronic stressors contribute differentially to the development of cardiovascular diseases involving blood pressure, vessel reactivity, and stress hormones. To investigate these mechanisms, we used restraint (acute ‐ 1 hr) and colony environmental housing (chronic ‐ 3 months) to examine changes in: systolic blood pressure (SBP), vessel reactivity to intraluminal pressure increases (myogenic reactivity, MYO), and plasma norepinephrine (NE) as an index for sympathetic nervous system (SNS) activity. We hypothesized that acute restraint stress will increase SBP, MYO of resistance‐sized mesenteric arteries, and plasma NE more compared to chronic environmental stress. Age‐matched adult SHR males were subjected to weekly restraint stress (n=7; 3 days/wk ‐ 1 month) or continuous colony housing stress (n=9) as compared to non‐stressed controls (n=7) in standard caging. Blood pressure was measured weekly via tail cuff, mesenteric artery reactivity was determined using a pressurized arteriograph, and plasma NE was assayed by HPLC. Acute restraint stressed animals exhibited increased MYO of mesenteric arteries compared to chronically stressed and control animals (p<0.05) possibly due to physiological adaptation in the colony housed rats. In conclusion, acute restraint stress may lead to increased SNS activity that results in increased MYO of resistance vessels involved in regulating blood pressure.

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.

Human cardiovascular diseases are often preceded by and exaggerated with stress, affecting blood pressure, catecholamines (CATS), and vessel reactivity. To investigate these mechanisms we used acute restraint stress and measured changes in: systolic blood pressure (SBP), plasma and urinary CATS, and responses of isolated renal interlobar arteries to the vasoconstrictor phenylephrine (PE). We hypothesized that stress would increase sympathetic nervous system (SNS) response by elevating SBP, both plasma and urinary CATS, and increase constriction of renal interlobar arteries. Adult male SHR rats were subjected to 4 weeks of 1hr restraint stress (n=8, 3dys/wk) compared to non‐stressed SHR (n=6) and WKY (n=6) controls. Data pending for stressed WKY males. Blood pressure was measured weekly via tail cuff, and CATS were assayed by HPLC. Small renal interlobar artery constriction of response to PE was determined using a pressurized arteriograph system. Stressed SHR animals exhibited increased plasma and urinary CATS. Constrictor responses to PE in stressed animals were decreased when compared to control animals, for example at 1.0mmol PE stressed SHRs demonstrated 48% maximum constriction while controls showed 80.3% (SHR) and 75.3% (WKY). In conclusion, acute stress does increase SNS activity through plasma CATS, but did not increase SBP. Vasoconstrictor responses of isolated arteries appear to be increased.

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.