Ovary-intact Females Research Articles

Sex-dependent remodeling of right ventricular function in a rat model of pulmonary arterial hypertension.

Right ventricular (RV) function is an important prognostic indicator for pulmonary arterial hypertension (PAH), a vasculopathy that primarily and disproportionally affects women with distinct pre- and postmenopausal clinical outcomes. However, most animal studies have overlooked the impact of sex and ovarian hormones on RV remodeling in PAH. Here, we combined invasive measurements of RV hemodynamics and morphology with computational models of RV biomechanics in sugen-hypoxia (SuHx)-treated male, ovary-intact female, and ovariectomized female rats. Despite similar pressure overload levels, SuHx induced increases in end-diastolic elastance and passive myocardial stiffening, notably in male SuHx animals, corresponding to elevated diastolic intracellular calcium. Increases in end-systolic chamber elastance were largely explained by myocardial hypertrophy in male and ovary-intact female rats, whereas ovariectomized females exhibited contractility recruitment via calcium transient augmentation. Ovary-intact female rats primarily responded with hypertrophy, showing fewer myocardial mechanical alterations and less stiffening. These findings highlight sex-related RV remodeling differences in rats, affecting systolic and diastolic RV function in PAH.NEW & NOTEWORTHY Combining hemodynamic and morphological measurements from male, female, and ovariectomized female pulmonary arterial hypertension (PAH) rats revealed distinct adaptation mechanisms despite similar pressure overload. Males showed the most diastolic stiffening. Ovariectomized females had enhanced myocyte contractility and calcium transient upregulation. Ovary-intact females primarily responded with hypertrophy, experiencing milder passive myocardial stiffening and no changes in myocyte shortening. These findings suggest potential sex-specific pathways in right ventricular (RV) adaptation to PAH, with implications for targeted interventions.

The aging ovary impairs acute stroke outcomes

In experimental stroke, ovariectomized (OVX) adult rats have larger infarct volumes and greater sensory-motor impairment as compared to ovary-intact females and is usually interpreted to indicate that ovarian hormones are neuroprotective for stroke. Previous work from our lab shows that middle-aged, acyclic reproductively senescent (RS) females have worse stroke outcomes as compared to adult (normally cycling) females. We hypothesized that if loss of ovarian estrogen is the critical determinant of stroke outcomes, then ovary-intact middle-aged acyclic females, who have reduced levels of estradiol, should have similar stroke outcomes as age-matched OVX. Instead, the data demonstrated that OVX RS animals showed better sensory-motor function after stroke and reduced infarct volume as compared to ovary-intact females. Inflammatory cytokines were decreased in the aging ovary after stroke as compared to non-stroke shams, which led to the hypothesis that immune cells may be extravasated from the ovaries post-stroke. Flow cytometry indicated reduced overall T cell populations in the aging ovary after middle cerebral artery occlusion (MCAo), with a paradoxical increase in regulatory T cells (Tregs) and M2-like macrophages. Moreover, in the brain, OVX RS animals showed increased Tregs, increased M2-like macrophages, and increased MHC II + cells as compared to intact RS animals, which have all been shown to be correlated with better prognosis after stroke. Depletion of ovary-resident immune cells after stroke suggests that there may be an exaggerated response to ischemia and possible increased burden of the inflammatory response via extravasation of these cells into circulation. Increased anti-inflammatory cells in the brain of OVX RS animals further supports this hypothesis. These data suggest that stroke severity in aging females may be exacerbated by the aging ovary and underscore the need to assess immunological changes in this organ after stroke.

Sexual dimorphism in the antigen-specific T cell response in S. Typhimurium infections within extra-lymphoid tissues

Abstract Differences in susceptibility to and severity of infectious diseases between the sexes are well established. Females often generate a beneficially stronger immune response when combatting infection but show an increased risk of developing autoimmunity. While the sex-related hormone estradiol is known to affect CD4 T cell cytokine release in vitro, less is known about T cells behavior in vivo. To address this, we intravenously infected male and female mice with attenuated Salmonella Typhimurium (S. Tm). By day 15 post infection (p.i.), male mice showed significantly lower rates of survival. Males also generated a higher number of S. Tm-specific CD4 T cells in the liver but, conversely, displayed higher bacterial burdens. To isolate the role of sex hormones, mice were gonadectomized before reaching sexual maturity and then infected. Ovariectomized mice lost significantly more weight than ovary-intact females by day 15 p.i. Like normal males, ovariectomized females demonstrated a significantly higher number of S. Tm-specific CD4 T cells but also higher liver bacterial burdens. To investigate the role of extra-lymphoid tissues such as the liver, we infected lymphotoxin-alpha (LTa) mice that genetically lack lymphoid tissues. Surprisingly, we found the stark difference in survival and CD4 T cell expansion between the sexes was lost. These differences in T cell expansion, and the commensurate effect on survival, in the presence or absence of endogenous sex hormones or lymphoid tissues indicate that females may have evolved a separate ability to regulate immune responses outside of traditional lymphoid organs. These studies will provide insights into sex-based differences seen in cellular immunity against bacterial pathogens in vivo. Supported by a grant from the Keck Foundation

Sexual dimorphism in vascular ATP-sensitive K+ channel function supporting interstitial via convective and/or diffusive O2 transport.

Inhibition of pancreatic ATP-sensitive K+ (KATP ) channels is the intended effect of oral sulphonylureas to increase insulin release in diabetes. However, pertinent to off-target effects of sulphonylurea medication, sex differences in cardiac KATP channel function exist, whereas potential sex differences in vascular KATP channel function remain unknown. In the present study, we assessed vascular KATP channel function (topical glibenclamide superfused onto fast-twitch oxidative skeletal muscle) supporting blood flow and interstitial O2 delivery-utilization matching ( is) during twitch contractions in male, female during pro-oestrus and ovariectomized female (F+OVX) rats. Glibenclamide decreased blood flow (convective O2 transport) and interstitial in male and female, but not F+OVX, rats. Compared to males, females also demonstrated impaired diffusive O2 transport and a faster fall in interstitial . Our demonstration, in rats, that sex differences in vascular KATP channel function exist support the tentative hypothesis that oral sulphonylureas may exacerbate exercise intolerance and morbidity, especially in premenopausal females. Vascular ATP-sensitive K+ (KATP ) channels support skeletal muscle blood flow ( ), interstitial O2 delivery ( )-utilization ( ) matching (i.e. interstitial-myocyte O2 flux driving pressure; is) and exercise tolerance. Potential sex differences in skeletal muscle vascular KATP channel function remain largely unexplored. We hypothesized that local skeletal muscle KATP channel inhibition via glibenclamide superfusion (5mg kg-1 GLI; sulphonylurea diabetes medication) in anaesthetized female Sprague-Dawley rats, compared to males, would demonstrate greater reductions in contracting (1Hz, 7V, 180s) fast-twitch oxidative mixed gastrocnemius (97% type IIA+IID/X+IIB) (15μm microspheres) and is (phosphorescence quenching), resulting from more compromised convective ( ) and diffusive ( ) O2 conductances. Furthermore, these GLI-induced reductions in ovary-intact females measured during pro-oestrus would be diminished following ovariectomy (F+OVX). GLI similarly impaired mixed gastrocnemius in both males (↓28%) and females (↓33%, both P<0.032) via reduced (male: ↓31%, female: ↓35%, both P<0.020), (male: 5.6±0.5 vs. 4.0±0.5, female: 6.4±1.1 vs. 4.2±0.6mL O2 min-1 100g tissue-1 , P<0.022) and the resulting is, with females also demonstrating a reduced (0.40±0.07 vs. 0.30±0.04mL O2 min-1 100g tissue-1 , P<0.042) and a greater GLI-induced speeding of is fall (mean response time: Sex×Drug interaction, P=0.026). Conversely, GLI did not impair the mixed gastrocnemius of F+OVX rats. Therefore, in patients taking sulphonylureas, these results support the potential for impaired vascular KATP channel function to compromise muscle and therefore exercise tolerance. Such an effect, if present, would likely contribute to adverse cardiovascular events in premenopausal females more than males.

Role for ovarian hormones in purinoceptor-dependent natriuresis

BackgroundPremenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats.MethodsTo test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750 μg/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1δ and ENaCα) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000 nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors.ResultsAcute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3.ConclusionThese data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

Tamoxifen Blocks the Development of Motivational Features of an Addiction-Like Phenotype in Female Rats.

Women become addicted sooner after initiating cocaine use as compared to men. Preclinical studies reveal a similar vulnerability in females, with findings from ovariectomized rats suggesting that estradiol mediates the enhanced vulnerability. However, since ovariectomy depletes not only estradiol, but all ovarian hormones, its role in a physiological context is not clear. Thus, the goal of this study was to determine the role of estradiol in the development of an addiction-like phenotype in ovary-intact females treated chronically with the selective estrogen receptor (ER) modulator tamoxifen. We hypothesized that tamoxifen, by antagonizing ERs, would block the development of an addiction-like phenotype as defined by an enhanced motivation for cocaine (assessed under a progressive-ratio schedule), and a heightened vulnerability to relapse (assessed under an extinction/cue-induced reinstatement procedure). Effects were examined following extended access cocaine self-administration (24-h/day; 4-discrete trials/h; 1.5 mg/kg/infusion) and 14-days of abstinence, conditions optimized for inducing an addiction-like phenotype. As predicted, motivation for cocaine was increased following extended-access self-administration and protracted abstinence in the vehicle (sesame oil) and no-injection control groups, but not in the tamoxifen group indicating that ER signaling is critical for the development of this feature of an addiction-like phenotype. Surprisingly, the increase in motivation for cocaine following abstinence was also attenuated in the vehicle group as compared to no-injection controls suggesting that oil/injections also affected its development. Contrary to our hypothesis, tamoxifen did not decrease vulnerability to relapse as this group responded at similar levels during initial extinction sessions and cue-induced reinstatement testing as compared to controls. Tamoxifen did, however, impair extinction learning as this group took longer to extinguish as compared to controls. Taken together, these findings indicate that estradiol is critical for the extinction of drug-associated cues and the development of motivational features of addiction.

Extraovarian gonadotropin negative feedback revealed by aromatase inhibition in female marmoset monkeys.

Whereas the ovary produces the majority of estradiol (E2) in mature female primates, extraovarian sources contribute to E2 synthesis and action, including the brain E2-regulating hypothalamic gonadotropin-releasing hormone. In ovary-intact female rodent models, aromatase inhibition (AI) induces a polycystic ovary syndrome-like hypergonadotropic hyperandrogenism due to absent E2-mediated negative feedback. To examine the role of extraovarian E2 on nonhuman primate gonadotropin regulation, the present study uses letrozole to elicit AI in adult female marmoset monkeys. Sixteen female marmosets (Callithrix jacchus; >2 yr) were randomly assigned to ovary-intact or ovariectomy (OVX) conditions and subsequently placed on a daily oral regimen of either ~200 µl vehicle alone (ovary-intact Control, n = 3; OVX, n = 3) or 1 mg ⋅ kg-1 ⋅ day-1 letrozole in vehicle (ovary-intact AI, n = 4; OVX + AI, n = 6). Blood samples were collected every 10 days, and plasma chorionic gonadotropin (CG) and steroid hormone levels were determined by validated radioimmunoassay and liquid chromatography/tandem mass spectrometry, respectively. Ovary-intact, AI-treated and OVX females exhibited elevated CG (P < 0.01, P = 0.004, respectively) compared with controls, and after 30 days, OVX + AI females exhibited a suprahypergonadotropic phenotype (P = 0.004) compared with ovary-intact + AI and OVX females. Androstenedione (P = 0.03) and testosterone (P = 0.05) were also elevated in ovary-intact, AI-treated females above all other groups. The current study thus confirms in a nonhuman primate that E2 depletion and diminished negative feedback in ovary-intact females engage hypergonadotropic hyperandrogenism. Additionally, we demonstrate that extraovarian estrogens, possibly neuroestrogens, contribute to female negative feedback regulation of gonadotropin release.

Estradiol alters the chemosensitive cardiac afferent reflex in female rats by augmenting sympathoinhibition and attenuating sympathoexcitation.

The chemosensitive cardiac vagal and sympathetic afferent reflexes are implicated in driving pathophysiological changes in sympathetic nerve activity (SNA) in cardiovascular disease states. This study investigated the impact of sex and ovarian hormones on the chemosensitive cardiac afferent reflex. Experiments were performed in anaesthetized, sinoaortic baroreceptor denervated male, female and ovariectomized female (OVX) Wistar rats with either intact cardiac innervation or bilateral vagotomy. To investigate the chemosensitive cardiac afferent reflexes renal SNA, heart rate (HR) and arterial pressure (AP) were recorded before and following application of capsaicin onto the epicardial surface of the left ventricle. Compared to males, ovary-intact females displayed similar cardiac afferent reflex mediated changes in renal SNA albeit with a reduced maximum sympathetic reflex driven increase in renal SNA. In females, ovariectomy significantly attenuated the cardiac vagal afferent reflex mediated inhibition of renal SNA (renal SNA decreased 2 ± 17% in OVX versus -50 ± 4% in ovary-intact females, P < 0.05) and augmented cardiac sympathetic afferent reflex mediated sympathoexcitation (renal SNA increased 91 ± 11% in OVX vs 62 ± 9% in ovary-intact females, P < 0.05) so that overall increases in reflex driven sympathoexcitation were significantly enhanced. Chronic estradiol replacement, but not progesterone replacement, begun at time of ovariectomy restored cardiac afferent reflex responses to be similar as ovary-intact females. Vagal denervation eliminated all group differences. The current findings show ovariectomy in female rats, mimicking menopause in women, results in greater chemosensitive cardiac afferent reflex driven sympathoexcitation and does so, at least partly, via the loss of estradiols actions on the cardiac vagal afferent reflex pathway.

Microglial activation occurs in the absence of anxiety-like behavior following microembolic stroke in female, but not male, rats.

BackgroundThe incidence of depression and anxiety disorders is twice as high in women than men; however, females exhibit less neuronal damage following an equivalent ischemic event. Microembolic stroke increases anxiety- and depressive-like behaviors in male rats but the behavioral repercussions in females are unknown.FindingsGiven the relative neuronal protection from stroke in ovary-intact females, female rats exposed to microembolic stroke may be behaviorally protected as compared to males. The data presented demonstrate that anxiety-like behavior is increased in males despite a comparable increase in microglial activation following microembolic stroke in both males and females.ConclusionsThese data suggest that males may be more behaviorally susceptible to the effects of microembolic stroke and further illustrate a dissociation between neuroinflammation and behavior in females.

Reflex control of renal sympathetic nerve activity in heart failure is preserved in ovary intact female rats (1171.3)

Following myocardial infarction (MI), pathological increases in sympathetic nerve activity (SNA) drive the development and progression of heart failure. The current study investigated the effects of female sex and ovarian hormones on arterial baroreflex and cardiac sympathetic afferent reflex (CSAR) control over renal SNA in MI‐induced heart failure. 6 weeks post MI or sham surgery , arterial pressure and renal SNA responses were recorded in chloralose‐urethane anesthetized male, female and ovariectomized female (OVX) rats. In male and OVX MI groups, arterial baroreflex control of renal SNA was significantly impaired compared to shams, due mostly to an impaired ability to inhibit SNA. In ovary‐intact females, arterial baroreflex control of renal SNA was unaltered in MI animals compared to sham. CSAR mediated increases in renal SNA were significantly augmented in male and OVX MI groups when compared to ovary‐intact female MI group. The current study is the first to describe a protective effect of ovarian hormones on reflex control of SNA in MI‐induced heart failure.Grant Funding Source: Lottery Health New Zealand and University of Auckland Faculty Research Development Fund

288 EFFECTS OF OVARIAN HORMONES ON THE CARDIAC AFFERENT REFLEX

Background: In disease states, such as hypertension and heart failure, the cardiac afferent reflex has been implicated in driving pathophysiological increases in sympathetic nerve activity (SNA). The current study set out to investigate the impact that ovarian hormones have on the cardiac afferent reflex. Methods: On the day of experiment blood pressure (BP), heart rate (HR) and renal SNA were recorded in chloralose-urethane anesthetized, open-chest and baroreceptor denervated female Wistar rats with ovaries-intact or -removed. The cardiac afferent reflex was investigated by placing capsaicin (1, 5 and 10 μg per 10 μl) via a piece of filter paper onto the anterior surface of the left ventricle for 40 seconds. Results: Removal of ovaries abolished the initial vagal mediated decrease in renal SNA that was observed in females with ovaries-intact at 4 seconds post capsaicin application. At 40 seconds post-capsaicin application the increase in renal SNA was significantly greater in ovary-removed females compared to ovary-intact females. The estrous cycle in ovary-intact females also significantly impacted on the cardiac afferent reflex; females in the proestrous phase displayed greater increases in renal SNA compared to females in the estrous/metestrous phase. Discussion: The current findings suggest that circulating ovarian hormones affect the cardiac afferent reflex, with removal of circulating ovarian hormones resulting in greater reflex sympathetic excitation. These findings provide evidence of a mechanism that may contribute to greater sympathetic excitation in cardiovascular disorders post-menopause, compared to pre-menopausal, which may relate to higher rates of morbidity and mortality in women following menopause.

Effects of sex and ovarian hormones on the initial renal sympathetic nerve activity response to myocardial infarction

The physiological mechanisms contributing to sex differences following myocardial infarction (MI) are poorly understood. Given the strong relationship between sympathetic nerve activity (SNA) and outcome, we hypothesized there may be a sex difference in SNA responses to MI. In anaesthetized, open-chest male, female and ovariectomized (OVX) female Wistar rats, mean arterial pressure, heart rate and renal SNA were recorded in response to ligation of the left coronary artery. In males, renal SNA increased by 30 ± 6% in the first minute of coronary occlusion (P < 0.05) and remained elevated at 18 ± 7% above baseline (P < 0.05) at 2 h following MI. In response to MI, ovary-intact females displayed no change in renal SNA, whereas OVX females displayed a significant increase, similar to that seen in the males (increases of 43 ± 11% at 1 min and 21 ± 7% at 2 h post-MI, P < 0.05 versus intact females). Arterial baroreflex control of renal SNA had a smaller range in females (ovary intact and OVX) than males; no changes in arterial baroreflex responses were observed 1 h post-MI in males or females. Denervating the arterial baroreceptors abolished the renal SNA response to MI in the males, whereas in ovary-intact females and OVX females the response was unaltered. These findings suggest that ovarian hormones are able to blunt the initial sympathetic activation post-MI in females and that the importance of the arterial baroreflex in mediating initial sympathetic activation post-MI is different between the sexes.

Developmental Programming: Contribution of Prenatal Androgen and Estrogen to Estradiol Feedback Systems and Periovulatory Hormonal Dynamics in Sheep1

Prenatal testosterone excess leads to neuroendocrine and periovulatory disruptions in the offspring culminating in progressive loss of cyclicity. It is unknown whether the mediary of these disruptions is androgen or estrogen, because testosterone can be aromatized to estrogen. Taking a reproductive life span approach of studying control, prenatal testosterone, and dihydrotestosterone-treated offspring, this study tested the hypothesis that disruptions in estradiol-negative but not -positive feedback effects are programmed by androgenic actions of testosterone and that these disruptions in turn will have an impact on the periovulatory hormonal dynamics. The approach was to test estradiol-negative and -positive feedback responses of all three groups of ovary-intact females during prepubertal age and then compare the periovulatory dynamics of luteinizing hormone, follicle-stimulating hormone, estradiol, and progesterone during the first breeding season. The findings show that estradiol-negative but not estradiol-positive feedback disruptions in prenatal testosterone-treated females are programmed by androgenic actions of prenatal testosterone excess and that follicular phase estradiol and gonadotropins surge disruptions during reproductive life are consistent with estrogenic programming. Additional studies carried out testing estradiol-positive feedback response over time found progressive deterioration of estradiol-positive feedback in prenatal testosterone-treated sheep until the time of puberty. Together, these findings provide insight into the mechanisms by which prenatal testosterone disrupts the reproductive axis. The findings may be of translational relevance since daughters of mothers with hyperandrogenism are at risk of increased exposure to androgens.

Thin layer chromatographic fraction of root extract of Polygonum hydropiper induces vaginal epithelial cell maturation in adult ovariectomized albino rat

Dry root powder of Polygonum hydropiper is traditionally used as an antifertility agent by women of north east India. The effect of crude root extract of P. hydropiper on reproductive performance of female albino rat was reported earlier from this laboratory. In the present investigation, attempt was made to find estrogenic effect, if any, of thin layer chromatographic fraction of the crude extract. To achieve the goal, methanolic crude extract was fractionated in thin layer chromatography (TLC). Estradiol-17β was used as the reference compound to identify the estrogenic compound in the crude root extract. The effect of TLC fraction was studied on the vaginal epithelial cell maturation in adult ovariectomized (OVX) rats and uterine histology of OVX and ovary-intact females. The TLC fraction was administered (sc) to adult OVX females at a dose of 5mg/kg/ day for 18 days. Estradiol-17β was injected as reference compound. Vaginal smears were stained with Giemsa's to observe the cell types. Rats were sacrificed by cervical dislocation at the end of the treatment and the uterine epithelium was subjected to histological analysis. The TLC fraction treated OVX females showed characteristic epithelial cell types (parabasal, intermediate, superficial and cornified) in the smear. Histological evidence revealed increased proliferation of the endometrial surface epithelium in the TLC fraction treated females. The results reveal that TLC fraction of root extract of Polygonum hydropiper contains compound(s) which has functional similarities with E2 and ovarian estrogen in female albino rats.

Estrogen promotes tumor progression in a genetically defined mouse model of lung adenocarcinoma

Numerous epidemiological observations point to sex differences in lung cancer etiology and progression. The present study was aimed at understanding the bases of these sex differences. To test the effect of estradiol on tumor progression, we used a mouse model based on conditional Kras expression and concurrent deletion of Tp53 following inhalation of an adenoviral vector expressing Cre recombinase (AdeCre). Ovariectomized females and males were treated with estradiol via a continuous-release capsule. Tumor multiplicity, tumor volume, and histological grade were determined at 10 weeks after AdeCre administration. Cell proliferation was monitored by Ki67 immunohistochemistry at 4 and 10 weeks after AdeCre administration. At 10 weeks, female mice had more than twice the number of tumors evident on the surface of the lungs than male mice; ovariectomy eliminated this sex difference. The estrogen treatment significantly increased tumor number and volume in ovariectomized females and in males. Histological character of the tumors ranged from adenoma to adenocarcinoma. Ovary-intact females exhibited higher grade tumors than ovariectomized females or males. Progression to higher histological grade was stimulated by estrogen in male mice but not in ovariectomized females. At 10 weeks after AdeCre administration, tumor cell Ki67-labeling varied widely, precluding assessment of an estrogen effect; however, at 4 weeks, Ki67 labeling of lung parenchymal cells was increased 3.5-fold by estrogen. In conclusion, estrogen acts as a promoter for lung adenocarcinoma in a genetically defined lung cancer model; estrogen-induced cell proliferation in the oncogene-initiated cells is likely to play a role in this tumor promoter activity.

Both ovarian hormones estrogen and progesterone are necessary for hormonal mammary carcinogenesis in ovariectomized ACI rats

August-Copenhagen-Irish (ACI) rats are unique in that the ovary-intact females develop high incidence of mammary cancers induced solely by hormones upon prolonged exposure to high levels of estrogen alone. Studies have also shown that such prolonged exposure to high-dose estrogen results in human-like aneuploid mammary cancers in ovary-intact ACI rats. To determine the role of progesterone in mammary carcinogenesis, six-week-old intact and ovariectomized ACI rats were continuously exposed to low- and high-dose estrogen alone, progesterone alone, low-dose estrogen plus progesterone, and ovariectomized ACI rats with high-dose estrogen plus progesterone. Also, ovariectomized ACI rats were treated with high-dose estrogen plus progesterone plus testosterone to determine the role of the androgen, testosterone, if any, in hormonal mammary carcinogenesis. The results indicate that continuous exposure to high, but not low, concentrations of estrogen alone can induce mammary carcinogenesis in intact but not in ovariectomized rats. Mammary carcinogenesis in ovariectomized ACI rats requires continuous exposure to high concentrations of estrogen and progesterone. The addition of testosterone propionate does not affect tumor incidence in such rats. These results suggest that both ovarian hormones estrogen and progesterone are necessary for mammary carcinogenesis induced solely by hormones in ovariectomized ACI rats. Our results are in agreement with the Women's Health Initiative studies, where treatment of postmenopausal women with estrogen (ERT) alone did not increase the risk of breast cancer, but estrogen and progesterone (HRT) did.

Gonadotrophin‐Releasing Hormone (GnRH) Release in Marmosets I: In Vivo Measurement in Ovary‐Intact and Ovariectomised Females

In vivo hypothalamic gonadotrophin-releasing hormone (GnRH) release was characterised for the first time in a New World primate. A nonterminal and repeatable push-pull perfusion (PPP) technique reliably measured GnRH in conscious common marmoset monkeys. Nineteen adult females (n = 8 ovary-intact in the mid-follicular phase; n = 11 ovariectomised) were fitted with long-term cranial pedestals, and a push-pull cannula was temporarily placed in unique locations within the pituitary stalk-median eminence (S-ME) 2 days prior to each PPP session. Marmosets underwent 1-3 PPPs (32 PPPs in total) lasting up to 12 h. Plasma cortisol levels were not elevated in these habituated marmosets during PPP, and PPP did not disrupt ovulatory cyclicity or subsequent fertility in ovary-intact females. GnRH displayed an organised pattern of release, with pulses occurring every 50.0 +/- 2.6 min and lasting 25.4 +/- 1.3 min. GnRH pulse frequency was consistent within individual marmosets across multiple PPPs. GnRH mean concentration, baseline concentration and pulse amplitude varied predictably with anatomical location of the cannula tip within the S-ME. GnRH release increased characteristically in response to a norepinephrine infusion and decreased abruptly during the evening transition to lights off. Ovary-intact (mid-follicular phase) and ovariectomised marmosets did not differ significantly on any parameter of GnRH release. Overall, these results indicate that PPP can be used to reliably assess in vivo GnRH release in marmosets and will be a useful tool for future studies of reproductive neuroendocrinology in this small primate.

Estrogen deficiency decreases ischemic tolerance in the aged rat heart: roles of PKCδ, PKCε, Akt, and GSK3β

The mechanisms underlying the age-dependent reversal of female cardioprotection are poorly understood and complicated by findings that estrogen replacement is ineffective at reducing cardiovascular mortality in postmenopausal women. Although several protective signals have been identified in young animals, including PKC and Akt, how these signals are affected by age, estrogen deficiency, and ischemia-reperfusion (I/R) remains unknown. To determine the independent and combined effects of age and estrogen deficiency on I/R injury and downstream PKC-Akt signaling, adult and aged female F344 rats ( n = 12/age) with ovaries intact or ovariectomy (Ovx) were subjected to I/R using Langendorff perfusion (31-min global-ischemia). Changes in cytosolic (s), nuclear (n), mitochondrial (m) PKC (δ, ε) levels, and changes in total Akt and mGSK-3β phosphorylation after I/R were assessed by Western blot analysis. Senescence increased infarct size 50% in ovary-intact females ( P &lt; 0.05), whereas no differences in LV functional recovery or estradiol levels were observed. Ovx reduced functional recovery to a greater extent in aged compared with adult rats ( P &lt; 0.05). In aged (vs. adult), levels of m- and nPKC(-δ, -ε) were markedly decreased, whereas mGSK3β levels were increased ( P &lt; 0.05). Ovx led to greater levels of sPKC(-δ, -ε) independent of age ( P &lt; 0.05). I/R reduced p-Akt(Ser473) levels by 57% and increased mGSK-3β accumulation 1.77-fold ( P &lt; 0.05) in aged, ovary-intact females. These data suggest, for the first time, that estrogen alone cannot protect the aged female myocardium from I/R damage and that age- and estrogen-dependent alterations in PKC, Akt, and GSK-3β signaling may contribute to loss of ischemic tolerance.

Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates

To further study the role of GPR54 signaling in the onset of primate puberty, we used the monkey to examine the ability of kisspeptin-10 to elicit the release of gonadotropin-releasing hormone (GnRH) precociously, and we describe the expression of GPR54 and KiSS-1 in the hypothalamus during the peripubertal period. Agonadal juvenile male monkeys were implanted with a lateral cerebroventricular cannula and a jugular vein catheter. The responsiveness of the juvenile pituitary to endogenous GnRH release was heightened with a chronic pulsatile i.v. infusion of synthetic GnRH before kisspeptin-10 (112-121) injection. Intracerebroventricular (30 microg or 100 microg) or i.v. (100 microg) bolus injections of kisspeptin-10 elicited a robust GnRH discharge, as reflected by luteinizing hormone secretion, which was abolished by pretreatment with a GnRH-receptor antagonist. RNA was isolated from the hypothalamus of agonadal males before (juvenile) and after (pubertal) the pubertal resurgence of pulsatile GnRH release and from juvenile, early pubertal, and midpubertal ovary-intact females. KiSS-1 mRNA levels detected by real-time PCR increased with puberty in both male and female monkeys. In intact females, but not in agonadal males, GPR54 mRNA levels in the hypothalamus increased approximately 3-fold from the juvenile to midpubertal stage. Hybridization histochemistry indicated robust KiSS-1 and GPR54 mRNA expression in the region of the arcuate nucleus. These findings are consistent with the hypothesis that GPR54 signaling by its cognate ligand in the primate hypothalamus may be activated at the end of the juvenile phase of development and may contribute to the pubertal resurgence of pulsatile GnRH release, the central drive for puberty.

Endocrine status affects bladder size and postvoid residual urinary volume in mice

Urine is one of the major media for intraspecific chemical communication in mice. The urination pattern is dependent both on the mice's hormonal and social status. The urination pattern and the morphology of the urinary tract were examined in mice following hormonal manipulations. In the first experiment, we compared pairs of intact and castrated males: intact males urinated earlier when exposed to a new environment, with a greater number of drops that were smaller than those of castrated males. In the second experiment, groups of intact males, castrated, testosterone-supplemented castrated, and isolated intact males were compared. The micturition pattern of isolated intact males consisted of numerous small droplets of urine, with a high volume of urine retained in the bladder after voiding. This also applied to grouped intact males and testosterone-treated castrated mice, while castrated mice voided a larger fraction of bladder content. Bladder weight was higher in intact males and testosterone-treated castrated males, as compared to castrated males. In the third experiment, ovary-intact and testosterone-treated intact females were compared. Testosterone-treated ovary-intact females retained a larger quantity of urine in the bladder and also had a larger bladder compared to ovary-intact females. Testosterone thus induces the morphological modifications of the urinary tract necessary for the dominant male urination pattern, which is an increase in postvoid urinary residual volume and bladder weight. As evidenced from the comparison of histological sections from intact, castrated, and testosterone-treated castrated males, the increase in bladder weight was mainly due to the bladder muscular mass.