Estrogen is formed by the enzyme aromatase (CYP19A1) and signals via three identified receptors ERα (ESR1), ERß (ESR2), and the G protein-coupled estrogen receptor (GPER). Understanding the relative contribution of each receptor to estrogenic signaling may elucidate the disparate effects of this sex hormone across tissues, and recent developments in PCR technology allow absolute quantification and direct comparison of multiple targets. We hypothesized that this approach would reveal tissue- and sex-specific differences in estrogen receptor mRNA. ESR1, ESR2, GPER, and CYP19A1 were measured in four cardiovascular tissues (heart, aorta, kidney, and adrenal gland), three brain areas (somatosensory cortex, hippocampus, and prefrontal cortex), and reproductive tissues (ovaries, mammary gland, uterus, testes) from six male and six female adult Sprague-Dawley rats. GPER mRNA expression was relatively stable across all tissues in both sexes, ranging from 5.49 to 113 copies/ng RNA, a 21-fold difference. In contrast, ESR1/ESR2 were variable across tissues although similar within an organ system. ESR1 ranged from 4.46 to 614 copies/ng RNA (138-fold difference) while ESR2 ranged from 0.154 to 83.1 copies/ng RNA (540-fold). Significant sex differences were broadly absent except for renal ESR1 (female 206 vs. male 614 copies/ng RNA, P < 0.0001) and GPER (62.0 vs. 30.2 copies/ng RNA, P < 0.05) as well as gonadal GPER (5.49 vs. 47.5 copies/ng RNA, P < 0.01), ESR2 (83.1 vs. 0.299 copies/ng RNA, P < 0.01), and CYP19A1 (322 vs. 7.18 copies/ng RNA, P < 0.01). Cardiovascular tissues showed a predominance of ESR1, followed by GPER. In contrast, GPER was the predominant transcript in the brain with similarly low levels of ESR1 and ESR2. CYP19A1 was detected at very low levels except for reproductive tissues and the hippocampus. While the data indicates a lack of sex differences in most tissues, significant differences were found in the range of receptor gene expression across tissues as well as in the receptor profile between organ systems. The data provide a guide for future studies by establishing estrogen receptor expression across multiple tissues using absolute PCR quantification. This knowledge on tissue-specific estrogen receptor profiles will aid the development of hormonal therapies that elicit beneficial effects in specific tissues.

Objective: A new strategy for menopausal hormone therapy replaces medroxyprogesterone with the selective estrogen receptor modulator bazedoxifene. While the agonist or antagonist activity of bazedoxifene has been examined in other tissues, the current study explored the impact of bazedoxifene on resistance artery reactivity. We hypothesized that bazedoxifene may induce greater vasoprotective effects than estradiol due to enhanced activation of the G-protein-coupled estrogen receptor. Methods: We measured the vasodilation of mesenteric resistance arteries from adult male and female wild-type and G-protein-coupled estrogen receptor knockout mice (n = 58) in response to increasing concentrations of bazedoxifene, medroxyprogesterone, and estradiol, and also the impact of these compounds on the responses to phenylephrine and sodium nitroprusside. Results: Bazedoxifene-induced vasorelaxation was greater than estradiol and blunted phenylephrine-induced contraction—an effect not observed with estradiol. Neither estradiol nor bazedoxifene altered relaxation to sodium nitroprusside. The combination of bazedoxifene + estradiol promoted greater vasodilation than medroxyprogesterone + estradiol, and opposed phenylephrine-induced contraction, whereas medroxyprogesterone + estradiol failed to attenuate this response. Both bazedoxifene + estradiol and medroxyprogesterone + estradiol enhanced sodium nitroprusside-induced relaxation in females. Vascular responses were similar in both sexes in wild-type and G-protein-coupled estrogen receptor knockout mice. Conclusion: Bazedoxifene and bazedoxifene + estradiol relaxed mesenteric arteries and opposed vasoconstriction to a greater degree than estradiol or medroxyprogesterone + estradiol. These effects were independent of sex and G-protein-coupled estrogen receptor expression. We conclude that bazedoxifene may provide vascular benefits over estrogen alone or estrogen plus progestogen combinations in postmenopausal women.

Objective: Our laboratory previously published that long-term administration of estradiol (E₂) was detrimental to the kidneys of midlife ovariectomized Long Evans rats, contrasting clinical studies in showing that menopausal hormone therapy is associated with decreased albuminuria. However, it is unknown whether this renal benefit was due to estrogen and/or the combination with progestogen. Therefore, the objective of the current study was to determine the impact of medroxyprogesterone (MPA) on E₂-mediated renal damage using a rodent model. Methods: Female Long Evans retired breeders underwent ovariectomy at 11 months of age and were treated for 40 days with subcutaneous E₂, E₂+MPA or vehicle at doses mimicking that of menopausal hormone therapy (N = 5-7 per group). Systolic blood pressure was measured along with indices of renal damage and function to investigate the impact of MPA on E₂-mediated renal outcomes. Renal estrogen receptor alpha and G protein-coupled estrogen receptor transcript copy numbers were measured in all treatment groups through droplet digital PCR. Results: Middle-aged female Long Evans rats displayed spontaneous hypertension with similar systolic blood pressures and heart weights between groups. Even though blood pressure was comparable, E₂ reduced glomerular filtration rate and increased proteinuria indicating pressure-independent renal damage. Coadministration with MPA prevented E₂-induced glomerular filtration rate impairment and proteinuria by promoting renal hypertrophy and preventing renal interstitial fibrosis. Both E₂ and E₂+MPA reduced renal estrogen receptor alpha (ERα) and increased renal G protein-coupled estrogen receptor mRNA, but neither ERα nor ERß protein was different between groups. Conclusion: MPA was protective against E₂-induced renal damage and dysfunction in middle-aged female Long Evans rats. Assessing the impact of hormone therapy on renal outcomes may be an important clinical factor when considering treatment options for postmenopausal women.

Arterial stiffness independently predicts cardiovascular mortality, coronary events, and stroke in hypertensive subjects and is exacerbated in women following menopause. Previously, our laboratory indicated that G protein-coupled estrogen receptor (GPER) plays a protective role in the vasculature. Therefore, the current study assessed the impact of sex and GPER on arterial stiffening in control and hypertensive conditions. We hypothesized that genetic deletion of GPER attenuates sex differences in arterial stiffness. Male and female wildtype (wt) and global GPER knockout (ko) mice (n=46) were used between 16-21 weeks of age. Angiotensin II (Ang II) infusion (700 ng/kg/day for two weeks) was used to induce hypertension, and systolic blood pressure (SBP) was measured using tail cuff plethysmography. Local pulse wave velocity (PWV) within the carotid artery was obtained via high frequency ultrasound in both color Doppler and M-mode. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. Baseline SBP was significantly lower in females (P=0.035) but was not impacted by genotype (P=0.78). Baseline PWV was significantly higher in male versus female wt mice (1.29 vs. 0.804 m/s, P=0.003) but was not different in ko mice (1.25 vs. 1.04 m/s, P=0.22). Ang II infusion significantly increased SBP (P<0.001) and PWV (P<0.001) in all groups, removing any impact of sex or genotype. In addition, significant correlations were found between Doppler and M-mode methods for obtaining carotid PWV (r=0.67, P<0.001) and between PWV and ex vivo carotid wall thickness (r=0.70, P=0.004). In contrast, SBP did not correlate with PWV (P=0.77) or carotid wall thickness (P=0.68). In conclusion, we found that GPER deletion did not impact blood pressure either in normotensive or hypertensive conditions. While arteries from female wt mice were less stiff at baseline, genetic deletion of GPER or Ang II infusion removed this protection independent of blood pressure. Moreover, we found that local carotid PWV provides information on vascular status that could not be obtained via blood pressure. This data indicates that GPER plays an important role in female vascular physiology that is absent in pathological conditions.