Y Are Males So Difficult to Understand?

Testosterone Can Stimulate Both Vasodilatory and Vasoconstrictory Pathways

The evidence of decreased arterial pressure in castrated SHRs, Dahl salt-sensitive, and Goldblatt hypertensive males compared with intact males suggests that testosterone influences arterial pressure regulation in diseased states.^24–31 Furthermore, elegant studies by Reckelhoff et al³⁰ demonstrated that testosterone treatment of ovariectomized female SHRs results in increased arterial pressure, suggesting that testosterone is a prohypertensive stimulus.

Interestingly, testosterone can stimulate both vasodilatory and vasoconstrictory pathways. In terms of vascular tone, testosterone causes an increase in sympathetic nervous system activity via an increase in tyrosine hydroxylase (leading to an increase in circulating noradrenaline levels)^32,33 and an increased release of 2 potent vasoconstrictors, angiotensin II (via upregulation of angiotensinogen production and renin activity^34–37) and neuropeptide Y.³⁸ Studies have demonstrated that the acute effects of testosterone are largely vasodilatory and are not mediated by conversion to estrogen or by activation of the androgen receptor.³⁹ It is interesting to note that the gene encoding the androgen receptor is located on the X chromosome.⁴⁰ It is suggested that testosterone-induced vasodilation may occur via regulation of potassium channels and not via activation of NO or regulation of calcium channels.^39,41 Testosterone has also been shown to promote atherosclerosis by stimulating changes in lipid profile (increasing low-density lipoproteins and decreasing high density lipoproteins),⁴² as well as alterations in vascular cell adhesion molecule 1,⁴³ therefore increasing monocyte adhesion to the vessel wall in the early stages of plaque formation.

The role of the kidney in blood pressure regulation has been elegantly highlighted using renal transplantation studies, which demonstrated that normotensive rats that received a hypertensive kidney transplant developed hypertension and hypertensive rats that received a normotensive kidney transplant exhibited decreased arterial pressure compared with hypertensive rats.⁴⁴ Consistent with the human condition, male SHRs have significantly higher arterial pressure than age-matched female SHRs. Given this difference in blood pressure between male and female SHRs, similar renal transplantation studies have also been performed in hypertensive rats to examine the role of the sex hormones. Transplantation of male SHR kidneys into female SHR rats, however, does not result in increased blood pressure and the converse, transplantation of female SHR kidneys into male SHRs does not reduce blood pressure.⁴⁵ These data suggests that, in the hypertensive rat, the hormonal milieu contributes to blood pressure regulation to a greater extent than the nonhormonal effects of the kidney. Extending this work, Reckelhoff et al⁴⁶ further investigated the role of the androgen receptor in the pressor effects of testosterone and demonstrated that blockade of the androgen receptor significantly lowered mean arterial pressure. This evidence suggests that the pressor response to testosterone is mediated through the androgen receptor. Testosterone can also directly interact with the renin-angiotensin system (RAS), upregulating angiotensinogen production, renin activity, and angiotensin II type 1 receptor expression, thereby enhancing the vasoconstrictory arm of the RAS.^34–37 In fact, plasma renin activity has been shown to be higher in males compared with females and is significantly reduced after castration in animals and enhanced in postmenopausal women.^30,47 Furthermore, the increase in angiotensinogen production, renin activity, and angiotensin II results in a rightward shift of the pressure-natriuresis response in males, resulting in sodium and water retention.^48,49 In addition to acting on the RAS to stimulate sodium and water retention, testosterone has been shown to upregulate the epithelial sodium channel-α, leading to enhanced sodium and water retention in the kidney,⁵⁰ which further contributes to increased arterial pressure in the presence of testosterone.

However, the sex hormones do not entirely account for differences between the sexes. Epidemiological data have demonstrated that 30% to 50% of the variation in blood pressure between individuals is attributable to genetic factors.^50,51 Given the differences in arterial pressure between males and females and the obvious genetic difference in the sex chromosomes between the sexes, it is suggested from both human and animal studies that the Y chromosome plays an integral role in the development of hypertension in males.^17,18,52,53

Y Chromosome Can Influence Blood Pressure Regulation

The human Y chromosome is ≈58 Mb long and encodes 86 genes, which result in 23 distinct proteins.⁵⁴ The role of these genes in sex organ development,^55–57 testosterone production,^55–57 and fertility^58,59 has been determined previously in humans. However, the exact contribution of the Y chromosome in the development of hypertension remains largely unknown. What is increasingly evident is that, in terms of cardiovascular function, the Y chromosome has both nongenomic effects (via the actions of testosterone, which may be independent of genetic interactions or may indirectly influence gene expression and regulation) and direct genomic effects. These genomic effects have been highlighted by the use of highly specialized animal models, which have provided the opportunity to dissect the role of the Y chromosome as distinct from that of testosterone.

Given the fact that the Y chromosome contains a gene(s) that encodes for testes development and testosterone production, it would seem that the hormonal and genetic influences of the Y chromosome are intrinsically linked and are, therefore, difficult to investigate independently. The use of 2 novel animal models to investigate the influence of the Y chromosome and the sex hormones on CVD development has extended the previous limitations of experimental design. Both of these models aim to remove or control for the influence of hormonal status (the presence or absence of testosterone) to determine the role of the Y chromosome genes, and both have significantly advanced our understanding of the mechanisms underlying male development of CVD. These models are the four core genotype (FCG) mouse model (developed by de Vries et al⁶⁰) and the Y consomic rat model (developed using the normotensive Wistar Kyoto [WKY] and the SHR strains by Ely and Turner⁵² and later using stroke-prone SHR [SHRSP] and WKY strains by Negrin et al⁵³).

FCG Mouse Model

The FCG mouse model was created to address the hypothesis that the XX or XY genotype accounts for sex differences independent of the gonadal or hormonal status. This model is based on 2 key findings that the development of ovaries and production of estrogen are the default position during fetal development and occur in the absence of testosterone⁶¹ and that the Sry gene located on the Y chromosome encodes for testes development and testosterone production.^62,63 The FCG model was developed by crossing 2 mouse models, one in which the Sry gene had been spontaneously deleted from the Y chromosome (named Y⁻) with a strain in which the Sry gene had been inserted onto an autosome (Figure 1).⁶⁰ This cross results in the formation of XX and XY⁻ with Sry on an autosome (named XX^Sry and XY^−Sry listed as male according to gonadal phenotype, XXM and XYM), which develop testes and produce testosterone, and the formation of XX and XY⁻ lacking the Sry gene (XX and XY⁻ listed as female according to gonadal phenotype, XXF and XYF), which develop ovaries and produce estrogen (Figure 1).⁶⁰ The offspring produced (XYM, XXM, XYF, and XXF) are then examined using a 2 by 2 comparison, which accounts for differences in hormonal status (XXM + XYM versus XXF + XYF), as well as sex chromosome complement (XXM + XXF versus XYM + XYF). The advantage of this approach is that the Y chromosome genetic complement can be assessed in the presence and absence of testosterone. This provides the opportunity to dissect the contribution of the sex chromosome complement and has yielded novel evidence of the genetic contribution to sex differences independent of hormonal status.

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Figure 1.

Four core genotypes (FCG) mouse model illustrating the progeny that result from the breeding of a female XX with a male XY⁻Sry, which has the Sry gene located on an autosome (A). The offspring produced are, therefore, XY⁻ and XX females and XY^−Sry and XX^Sry males with sex (male or female) indicative of gonadal phenotype (adapted with permission from Ji et al⁶⁵).

To date, the majority of studies using the FCG model have investigated sex differences in behavior and neural function. Investigations include sex differences in aggression, parenting, social interaction, nociception, habit formation related to drug abuse, neural tube closure, multiple sclerosis, and systemic lupus erythematosus (for a comprehensive review of these investigations see Arnold et al⁶⁴). These studies have significantly advanced the field of sex differences in brain function and behavior. To our knowledge, this FCG model has only recently been used to study sex differences in blood pressure regulation and CVD, with the first article published in 2010.⁶⁵ Given the known sex differences in renal, cardiac, and vascular function and the pathophysiology of CVD, the FCG model has the potential to greatly advance our understanding of sex differences in blood pressure regulation and cardiovascular function.

Y Consomic Rat Model

In contrast, the Y consomic rat model approach was designed to specifically test the hypothesis that there are genes located on the Y chromosome that influence the development of CVD in the presence of testosterone. The consomic model involves the production of 2 strains of rat, one with the normotensive WKY autosomes and X chromosome in which the hypertensive SHR Y chromosome has been introgressed (WKY.SHR[Y]; Figure 2) and the other that has the hypertensive SHR autosomes and X chromosome with the normotensive WKY Y chromosome introgressed (SHR.WKY[Y]; Figure 2). The nomenclature of the consomic strains is as follows: the first abbreviation indicates the background strain (the origin of the autosomes and X chromosome), and the second abbreviation indicates the origin of the introgressed Y chromosome. Investigation of these consomic strains in parallel with their parental strains allows for the dissection of the “hypertensive” versus “normotensive” Y chromosome effects. The advantage of this approach is that all 4 of the strains are exposed to typical male cues (eg, testes development, testosterone production, and androgen receptors), suggesting that any differences observed are unlikely attributed to the effects of testosterone per se.

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Figure 2.

Y consomic rat breeding design and progeny. The top panel depicts the parental cross resulting in the generation of Wistar-Kyoto (WKY), spontaneously hypertensive rat (SHR), or the consomic cross with the WKY background strain and the Y chromosome from the SHR (WKY.SHR[Y]) or the SHR background and the Y chromosome from the WKY (SHR.WKY[Y]). The bottom panel illustrates the progeny that result from this breeding approach.

Limitations of the FCG Mouse Model and the Y Consomic Rat Model

As with all animal models, both have some limitations. The FCG model has 4 main restrictions. First, the FCG model can be used to determine the contribution of the sex chromosome complement but is unable to distinguish between the influence of the X chromosome compared with the Y chromosome. Second, the Sry gene has been shown recently to have functions independent of testes development and testosterone production,⁶⁶ which are difficult to dissect in the FCG model and complicate the interpretation of the experimental findings in this model. Third, the FCG model assumes that the genes located on the Y chromosome will have the same expression, regulation, and function in the absence of typical male cues (testes development, testosterone production, and androgen receptors) and/or the presence of female cues (ovary development and estrogen production). Last, the FCG model assumes that introgression of the Sry locus onto an autosome (as opposed to the original location on the Y chromosome) will not alter its activity and/or expression.

In contrast, the Y consomic rat model is unable to be used to determine the contribution of the X chromosome, because this model uses males that have the same number of X chromosomes. Furthermore, the Y consomic approach does not investigate the contribution of the Y chromosome in the absence of previous or continued testosterone stimulation. That is, even after castration, the Y consomic model cannot be used to directly answer the question as to whether the genes located on the Y chromosome have in some way been influenced by the presence of testes and testosterone before castration. Despite the obvious species difference between these models, what is particularly striking is that the limitations of one represent the strengths of the other, and as such we suggest that the findings of these 2 models should be interpreted together to provide the best insights into the mechanisms underlying sex differences in males. So, what have we learned about the role of Y chromosome in CVD from these models?

The Sex Chromosome Complement Influences Blood Pressure Regulation

In contrast to the human condition, sex differences in blood pressure have only been reported in animal models of disease, such as Goldblatt hypertensive rats, SHRs, Dahl-salt sensitive rats, and angiotensin II–induced hypertension. Therefore, investigation of the FCG model in the context of angiotensin II–induced hypertension provides a model with enhanced sex differences compared with the normotensive situation. Ji et al⁶⁵ used the FCG model to investigate the effect of the sex chromosomes on angiotensin II–induced hypertension. There was no difference in blood pressure before angiotensin II treatment between any of the genotypes (XXF, XYF, XXM, and XYM), which is not surprising, because all of the rats were gonadectomized before entering the study protocol. Ji et al⁶⁵ reported for the first time that sex chromosome complement significantly influenced blood pressure response to chronic angiotensin II infusion, with the XY genotype (XYM and XYF) having a blunted pressor response to angiotensin II infusion compared with the XX genotype (XXM and XXF). These data are surprising given the growing body of work demonstrating in both humans and animal models that females have a blunted pressor response to angiotensin II infusion compared with males.^15,67–72 It is tempting to speculate that the sex hormones and the sex chromosome complement may in fact have opposite effects and the sum of these effects may determine the net outcome, a hypothesis that will become clearer when understanding of the role of the X and Y chromosomes draws closer to, or even matches, the understanding of the sex hormones. Conversely, gonadal sex did not contribute to blood pressure responses to chronic angiotensin II infusion, but there were significant differences in heart rate depending on gonadal sex, with the female phenotype demonstrating a higher heart rate than the male phenotype.⁶⁵ These data provide the first evidence that the sex chromosome complement (XX or XY) plays an important role in blood pressure regulation irrespective of gonadal sex. As mentioned previously, studies investigating the FCG model unfortunately cannot dissect the influence of the X versus Y chromosome. In this instance, the studies conducted in the Y consomic model extend the work conducted in the FCG model.

The Y Chromosome Accounts for 12 to 15 mm Hg of Systolic Blood Pressure

Ely et al⁵² were the first to demonstrate the influence of the Y chromosome on blood pressure regulation. Introgression of the hypertensive Y chromosome from the SHR into the WKY background strain resulted in an increase in systolic blood pressure of 12 mm Hg with the introgression of the normotensive WKY Y chromosome into the SHR, resulting in a decrease in systolic blood pressure of ≈14 mm Hg compared with the SHR.⁵² This finding was confirmed using a similar Y consomic approach in the SHRSP and WKY rat strains originating from the Glasgow colony.⁵³ Negrin et al⁵³ observed higher arterial pressure in the WKY.SP_GlaY compared with the WKY (15 mm Hg higher), demonstrating that, in the WKY, arterial pressure level is influenced by the hypertensive Y chromosome. Arterial pressure responses to a high salt challenge were also shown to be Y chromosome dependent. Negrin et al⁵³ demonstrated that, after 2 weeks of 1% salt challenge, the SP.WKY_GlaY had lower systolic pressure compared with the SHRSP (30 mm Hg lower), despite the fact there was no significant difference in systolic pressure between SHRSP and SP.WKY_GlaY before the salt challenge. Taken together, these data demonstrates that the Y chromosome influences arterial pressure regulation and supports the hypothesis that there may be a hypertensive Y. The intriguing Y chromosome–dependent differences in response to salt treatment are in line with recent reports demonstrating that renal overexpression of the Sry3 gene, a gene that is a member of the Sry gene family and is located exclusively on the SHR Y chromosome, resulted in a 50% increase in renal sodium reabsorption in Sry3-overexpressed WKY rats compared with control WKY rats.⁷³ The authors suggest that this may be explained by an increase in renal angiotensin II, known to promote vasoconstriction and sodium reabsorption in the kidney.⁷³ They also observed that overexpression of Sry3 resulted in a decrease in glomerular filtration rate, which is of particular interest given the sex differences in the rates and progression of end-stage renal disease in humans, with men progressing at a faster rate than premenopausal women.^74–76

Members of the Sry Gene Family, Located on the Y Chromosome, Influence Arterial Pressure Regulation via Interaction With the Sympathetic Nervous System and the RAS

Ely and Turner et al,⁵² using the Y consomic rat approach, have demonstrated novel blood pressure regulation effects of the Sry gene family, independent of its role in gonad development. Turner et al⁶⁶ have provided compelling evidence supporting the prohypertensive effects of the Sry gene family, which is located in the nonrecombining region of the Y chromosome. In the rat there are multiple copies of the Sry gene, which is a highly conserved gene encoding for testes development and testosterone production (as described in the FCG mouse model).

The impetus to study the influence of the Sry genes in blood pressure regulation arose from the finding that the Sry gene family members are expressed not only in sex determining organs, such as the testes, but also in tissues involved in blood pressure regulation, such as the brain, kidney, and adrenal gland.^77–79 In the rat there are ≤7 copies of the Sry gene, Sry1, Sry2, Sry3, Sry3A, Sry3BI, Sry3B, and Sry3C.⁶⁶ Interestingly, all of the copies are found in the hypertensive SHR strain, with only 6 copies found in the WKY strain; Sry3 is found exclusively on the SHR Y chromosome. Ely and colleagues^73,80 have successfully sequenced the Sry copies and have determined, via renal overexpression studies using electroporation, that Sry1 and Sry3 elevate blood pressure by 10 to 20 mm Hg, whereas Sry2 does not influence blood pressure.⁷³ The increase in blood pressure by Sry1 overexpression is mediated via an increase in sympathetic nervous system activity, because Sry1 directly enhances tyrosine hydroxylase promoter activity⁸¹ and thereby increases noradrenaline levels. The concept of sympathetic nervous system involvement in blood pressure regulation is far from novel.^82–86 However, given the recent success of renal denervation in the treatment of essential hypertension,^87,88 a genetic mechanism underlying sympathetic nervous system overstimulation can be hypothesized, at least in males.

Conversely, Sry3 can interact with the RAS in vitro to upregulate angiotensinogen, renin, and ACE promoter activity and to decrease ACE2 promoter activity, suggesting a shift in the RAS toward enhanced vasoconstrictive responses in the presence of Sry3.⁸⁹ The in vivo functional consequences of Sry3 on the RAS are not yet fully elucidated; however, Ely et al⁷³ demonstrated recently that increased expression of Sry3 in vivo, achieved by electroporation of exogenous Sry3, resulted in a 40% increase in renal ACE concentration and concomitantly increased renal angiotensin II concentration. As with the function of all genes, determining the exact influence of these Sry copies in vivo will be complex. It is likely that the activity of these genes (in this case, the interaction of the Sry genes with other gene promoters, eg, angiotensinogen) in vivo will be significantly different to the in vitro responses. For example, previous work has demonstrated that the ACE promoter contains ≥2 shear stress response elements,⁹⁰ as well as 2 cyclic adenosine-monophosphate response elements,⁹¹ which also regulate promoter activity, in addition to the effects of the Sry3 gene demonstrated in vitro.⁷³ Hence, in the in vivo setting, the possible role of Sry3 to regulate ACE promoter activity may be masked by other factors also contributing to ACE promoter activity, particularly in the SHR and SHRSP hypertensive models, where shear stress is likely to be a main contributor. Nonetheless, the RAS plays a major role in the regulation of blood pressure, with a growing body of evidence demonstrating that the mean arterial pressure pressor response to angiotensin II is attenuated in females compared with males.^{15,69–72,92} As discussed previously, the sex hormones are known to interact directly with the RAS^49,93–95; however, the potential nonhormonal sex differences in the regulation of the RAS are less well defined. Recent work by Liu et al⁹⁶ reported that male mice have a higher renal ACE2 activity than female mice, perhaps suggesting a greater influence of the newly discovered vasodilatory arm of the RAS in males. The ACE2 gene is located on the X chromosome, which opens the possibility for sex differences in the expression levels between males and females dependent on the number of X chromosomes present (1 in males and 2 in females) and the complex nature of X chromosome inactivation. Liu et al⁹⁶ suggest that ACE2 activity is not sex chromosome dependent but rather estrogen dependent. Using the FCG mouse model, Liu et al⁹⁶ measured ACE2 activity in kidneys from intact, gonadectomized, and gonadectomized + estrogen-replaced mice and observed that estrogen replacement reduced ACE2 activity, irrespective of sex chromosome complement.⁹⁶ These data suggests that the sex chromosome complement does not influence the ACE2 expression in the FCG mouse model.⁹⁶

Future Directions, Conclusions, and Perspectives

Investigation of blood pressure regulation in the FCG mouse model and the Y consomic rat model has significantly advanced the understanding of the mechanisms underlying the sex differences in blood pressure observed across all species. Despite a number of well-designed, large-scale, genomewide association studies, the exact genes involved in arterial pressure regulation remain difficult to determine, much less the influence of these genes in the complex sex differences in blood pressure regulation. The promising findings using the FCG and Y consomic animal models provide compelling evidence of the contribution of the sex chromosome complement, in particular the Y chromosome in blood pressure regulation. By using these novel animal models to investigate the sex differences in blood pressure regulation and CVD, researchers are afforded the opportunity to elucidate the mechanisms underlying these sex differences that are observed across species and apply the findings to the study of human biology.