Search

ObjectiveHigh grade endometrial stromal sarcoma and undifferentiated uterine sarcomas are associated with a very poor prognosis. Although large surgical resection is the standard of care, the optimal adjuvant strategy remains unclear.MethodsA retrospective analysis of patients with localized high grade endometrial stromal sarcoma and undifferentiated uterine sarcomas (stages I–III) treated in 10 French Sarcoma Group centers was conducted.Results39 patients with localized high grade endometrial stromal sarcoma and undifferentiated uterine sarcomas treated from 2008 to 2016 were included. 24/39 patients (61.5%) were stage I at diagnosis. 38/39 patients underwent surgical resection, with total hysterectomy and bilateral oophorectomy completed in 26/38 (68%). Surgeries were mostly resection complete (R0, 23/38, 60%) and microscopically incomplete resection (R1, 6/38, 16%). 22 patients (58%) underwent postoperative radiotherapy (including brachytherapy in 11 cases), and 11 (29%) underwent adjuvant chemotherapy. After a median follow-up of 33 months (range 2.6–112), 17/39 patients were alive and 21/39 (54%) had relapsed (9 local relapses and 16 metastases). The 3 year and 5 year overall survival rates were 49.8% and 31.1%, respectively, and 3 year and 5 year disease free survival rates were 42.7% and 16.0%, respectively. Median overall survival and disease free survival were 32.7 (95% CI 16.3–49.1) and 23 (4.4–41.6) months, respectively. Medians were, respectively, 46.7 months and 39.0 months among those who underwent adjuvant radiotherapy and 41.0 months and 10.3 months for those who underwent adjuvant chemotherapy. In multivariate analysis, adjuvant radiotherapy was an independent prognostic factor for overall survival (P=0.012) and disease free survival (P=0.036). Chemotherapy, International Federation of Gynecology and Obstetrics I–II stages, and Eastern Cooperative Oncology Group-performance status 0 correlated with improved overall survival (P=0.034, P=0.002, P=0.006), and absence of vascular invasion (P=0.014) was associated with better disease free survival.ConclusionsThe standard treatment of primary localized high grade endometrial stromal sarcoma and undifferentiated uterine sarcomas is total hysterectomy and bilateral oophorectomy. The current study shows that adjuvant radiotherapy and adjuvant chemotherapy appear to improve overall survival. A prospective large study is warranted to validate this therapeutic management.

International audience; OBJECTIVE : The severity of rheumatoid arthritis (RA) correlates directly with bone erosions arising from osteoclast (OC) hyperactivity. Despite the fact that inflammation may be controlled in patients with RA, those in a state of sustained clinical remission or low disease activity may continue to accrue erosions, which supports the need for treatments that would be suitable for long-lasting inhibition of OC activity without altering the physiologic function of OCs in bone remodeling. Autotaxin (ATX) contributes to inflammation, but its role in bone erosion is unknown. METHODS : ATX was targeted by inhibitory treatment with pharmacologic drugs and also by conditional inactivation of the ATX gene Ennp2 in murine OCs (ΔATXC tsk ). Arthritic and erosive diseases were studied in human tumor necrosis factor-transgenic (hTNF+/- ) mice and mice with K/BxN serum transfer-induced arthritis. Systemic bone loss was also analyzed in mice with lipopolysaccharide (LPS)-induced inflammation and estrogen deprivation. Joint inflammation and bone erosion were assessed by histology and micro-computed tomography. The role of ATX in RA was also examined in OC differentiation and activity assays. RESULTS : OCs present at sites of inflammation overexpressed ATX. Pharmacologic inhibition of ATX in hTNF+/- mice, as compared to vehicle-treated controls, significantly mitigated focal bone erosion (36% decrease; P < 0.05) and systemic bone loss (43% decrease; P < 0.05), without affecting synovial inflammation. OC-derived ATX was revealed to be instrumental in OC bone resorptive activity and was up-regulated by the inflammation elicited in the presence of TNF or LPS. Specific loss of ATX in OCs from mice subjected to ovariectomy significantly protected against the systemic bone loss and erosion that had been induced with LPS and K/BxN serum treatments (30% reversal of systemic bone loss [P < 0.01]; 55% reversal of erosion [P < 0.001]), without conferring bone-protective properties. CONCLUSION : Our results identify ATX as a novel OC factor that specifically controls inflammation-induced bone erosions and systemic bone loss. Therefore, ATX inhibition offers a novel therapeutic approach for potentially preventing bone erosion in patients with RA.

Objective— Whereas estradiol prevents fatty streak deposit in immunocompetent apoE −/− or LDLr −/− mice, it is totally ineffective in immunodeficient mice, underlining the key role of immunoinflammation in this effect. In the present work, the role of several major pro- and antiinflammatory cytokines involved in the atheromatous process was evaluated in the effect of estradiol on fatty streak constitution. Methods and Results— The preventive effect of estradiol was fully maintained in LDLr −/− mice grafted with bone marrow from either IFN-γ or interleukin (IL)-12–deficient mice, showing that this beneficial effect was not mediated through a specific decrease in the production of these 2 proinflammatory cytokines. Furthermore, IL-10 −/− apoE −/− mice remained protected by estradiol, excluding a significant contribution of this antiinflammatory cytokine. In contrast, the protective effect of estradiol was (1) associated with enhanced aortic expression of TGF-β1 in apoE −/− mice during early steps of atherogenesis; (2) abolished and even reversed in apoE −/− mice administered with a neutralizing anti–TGF-β antibody; (3) abolished in LDLr −/− mice grafted with bone marrow from Smad3-deficient mice. Conclusions— The status of the TGF-β pathway crucially determines the antiatherogenic effect of estradiol in hypercholesterolemic mice, whereas neither IFN-γ, IL-12, nor IL-10 are specifically involved in this protection.

International audience; This study aims to investigate venous thromboembolism (VTE) risk in relation to age at menopause, age at menarche, parity, bilateral oophorectomy, and time since menopause, as well as any interaction with randomized hormone therapy (HT) assignment, among postmenopausal women. Using pooled data from the Women's Health Initiative HT clinical trials including 27,035 postmenopausal women aged 50 to 79 years who had no history of VTE, we assessed the risk of VTE in relation to age at menopause, age at menarche, parity, bilateral oophorectomy, and time since menopause by Cox proportional hazards models. Linear trends, quadratic relationships, and interactions of reproductive life characteristics with HT on VTE risk were systematically tested. During follow-up, 426 women reported a first VTE, including 294 non-procedure-related events. No apparent interaction of reproductive life characteristics with HT assignment on VTE risk was detected, and there was not a significant association between VTE and age at menarche, age at menopause, parity, oophorectomy, or time since menopause. However, analyses restricted to non-procedure-related VTE showed a U-shaped relationship between age at menopause and thrombotic risk that persisted after multivariable analysis (P < 0.01). Compared with women aged 40 to 49 years at menopause, those who had early menopause (age 55 y) had a significantly increased VTE risk (hazard ratio [95% CI]: 1.8 [1.2-2.7] and 1.5 [1.0-2.4], respectively). Reproductive life characteristics have little association with VTE and do not seem to influence the effect of HT on thrombotic risk among postmenopausal women. Nevertheless, early and late onset of menopause might be newly identified risk factors for non-procedure-related VTE.

Objective— To assess the coronary endothelial protective effects of 17β-estradiol (E2) and the role of estrogen receptor (ER) α in ischemia/reperfusion (I/R). Methods and Results— E2 exerts protective effects in cardiac I/R. However, the implication in vivo of the endothelium and the cellular targets of the anti-ischemic effects of E2 are unknown. Mice were subjected to I/R (30 minutes of I and 1 hour of R) in vivo, after which acetylcholine-induced relaxation of isolated coronary segments was assessed ex vivo. I/R induced a coronary endothelial dysfunction in untreated ovariectomized mice that was prevented by long-term treatment with E2 in wild-type, but not in ERα −/− , mice. Chimeric mice inactivated for ERα in the hematopoietic compartment remained protected by E2. Further inactivation of endothelial ERα abolished the protective action of E2 on coronary endothelial function in Tie2-Cre(+) ERα f/f mice. More importantly, E2 significantly limited infarct size in wild-type mice but not in mice deficient in endothelial ERα, even in the presence of hematopoietic ERα. Conclusion— Endothelial ERα plays a crucial role in the E2-induced prevention of endothelial dysfunction after I/R. To our knowledge, we demonstrate for the first time, by using unique genetically modified mice, that targeting endothelial protection per se can confer cardiomyocyte protection in I/R.

International audience; Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor α expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor α, exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.

Background— The prevention of early atheroma by estrogens has been clearly demonstrated in all animal models and appears to be mediated through a direct action on the arterial wall rather than through an effect on the lipoprotein profile. The goal of the present study was to evaluate which cellular target is crucial in this beneficial action of estradiol. Methods and Results— We first confirmed the key role of estrogen receptor-α (ERα) in the atheroprotective effect of estradiol, because this action was completely abolished in mice deficient in both the low-density lipoprotein receptor (LDLr) and ERα. Second, using chimeric mice with an ERα deficiency in the hematopoietic lineage, we showed the persistence of the protective action of estradiol, which suggests the involvement of extrahematopoietic ERα. Third, we showed that loxP-flanked ERα mice (ERα flox/flox ) bred with Tie2-Cre + mice on an LDLr −/− background had complete inactivation of ERα in most hematopoietic and all endothelial cells. Remarkably, in this mouse model, the atheroprotective effect of estradiol was completely abolished. Fourth, the atheroprotective effect of estradiol remained abolished in Tie2-Cre + ERα flox/flox LDLr −/− mice transplanted with either Tie2-Cre + ERα flox/flox or ERα −/− bone marrow, whereas it was present in analogous chimeric Tie2-Cre − ERα flox/flox LDLr −/− receivers expressing endothelial ERα. Conclusions— We demonstrate directly and for the first time that endothelial ERα represents a key target of the atheroprotective effect of estradiol, whereas hematopoietic ERα is dispensable. Selective estrogen receptor modulators that mimic the endothelial action of estradiol should now be considered in atheroprotection.

International audience; Lung tissue, both normal and cancerous, has been found to express estrogen receptors and patterns of expression have differed between men and women, suggesting a possible role for hormone-related factors in lung carcinogenesis in women. Few epidemiological studies have examined hormone-related variables and lung cancer risk and the findings have not been consistent. We investigated the association between characteristics of menstruation and pregnancy in relation to lung cancer risk in a population-based case-control study carried out in Montreal, Canada, including 422 women with lung cancer and 577 controls. For each variable, odds ratios (OR) and 95% confidence intervals (CI) were estimated using unconditional logistic regression modeling. Associations were also examined according to level of smoking and by lung cancer histology. All statistical tests were two-sided. Most characteristics of menstruation and pregnancy were not associated with lung cancer risk. However, an increased risk was observed for women who had had a non-natural menopause, which predominantly included women who had had a bilateral oophorectomy, compared with women who had had a natural menopause (OR = 1.92, 95% CI: 1.22-3.01). An inverse association with age at menopause was suggested. These results did not vary by level of smoking and they were similar for adenocarcinomas compared with other histological types. Our results suggest that hormonal factors, related to early menopause and/or ovary removal, may play a role in the risk of lung cancer. Further studies are needed to confirm these findings, and to assess the possible contribution of hormone replacement therapy.

International audience; OBJECTIVES: The ovarian remnant syndrome is a rare condition after unilateral or bilateral oophorectomy, with or without a hysterectomy. This syndrome occurs when a fragment of ovarian tissue is left behind and becomes functional and cystic. The purpose of this study is to report the cases of patients treated surgically for an ovarian remnant syndrome during the last 10 years and to recall the diagnostic and therapeutic difficulties. PATIENTS AND METHODS: A retrospective, observational study was carried out between 1997 and 2006. Seven patients were treated surgically for an ovarian remnant syndrome. Perioperative data analysis (history, surgical techniques, and postoperative follow-up) was carried out. RESULTS: The mean age of the patients was 46 years (36-55). The number of previous abdominal surgical procedures ranged from 2 to 5. The syndrome appeared after a mean period of 4 years and 4 months (range 5 months-12 years) after oophorectomy. Among the 7 patients, 3 had had a previous hysterectomy. Pelvic pain was found in all cases. Gonadotropin-releasing hormones agonists were used in 1 patient without success. Aspiration was performed in 2 cases before surgical treatment. Two patients underwent a laparotomy in the first place. Laparoscopy was performed in 5 cases and laparoconversion was necessary in 1 case. Intraoperative difficulties and anatomic variations were found in all cases. Ureteral catheters were placed in 2 cases. Radiotherapy was performed in 1 patient who had a recurrent ovarian remnant. DISCUSSION AND CONCLUSION: The ovarian remnant syndrome is a rare complication. Surgery, either by laparoscopy or by laparotomy, is the recommended treatment. These operations are often difficult and associated with a high risk of complications. Histologically, remnant ovarian tissue associated with hemorragic corpus luteum cysts is the most common finding. The prevention of the ovarian remnant syndrome is based on rigorous surgical treatment during the oophorectomy so as not to leave behind ovarian tissue.

Full-length 66-kDa estrogen receptor α (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERα AF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ER α AF-1 0 mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-week-old ER α AF-1 +/+ LDLr −/− (low-density lipoprotein receptor) and ER α AF-1 0 LDLr −/− mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the aortic tree of 8-month-old ovariectomized or intact female mice revealed that ERα AF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERα AF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

Although 17β-estradiol (E2) is protective in experimental models of myocardial and brain ischemia, its effect on skin ischemia remains unknown. Here, we assessed the protective effect of E2 in a mouse model of skin ischemia, mimicking the surgery of skin flaps. Whereas necrosis appeared in the half portion of the skin flap within 1 week after surgery in ovariectomized mice, it was reduced up to 10-fold when mice were pretreated with E2, at least 3 days before the surgery. The beneficial effect of E2 appeared to be attributable to an increase in skin survival, revealed by measuring viability of ex vivo explants and enhancement of the antiapoptotic Bcl-2 protein expression in vivo. This protective effect on the skin contributed to the protection of the vascular network and facilitated reperfusion, which was found to be accelerated in ovariectomized E2-treated mice, whereas hemorrhages were observed in untreated mice. E2 also increased expression of fibroblast growth factor-2 isoforms in the skin and circulating vascular endothelial growth factor in the serum. Finally, this protective effect of E2 was abolished in estrogen receptor–deficient mice (ERα −/− ) but maintained in chimeric mice reconstituted with ERα-deficient bone marrow, indicating dispensable action of E2 in bone marrow–derived cells. This protective effect of E2 was mimicked by treatment with tamoxifen, a selective estrogen receptor modulator. In conclusion, we have demonstrated for the first time that E2 exerts a major preventive effect of skin flap necrosis through a prevention of ischemic-induced skin lesions, including those of the vascular network, which contributes to accelerate the reperfusion of the skin flap.

International audience; The ultradian pulsatile pattern of growth hormone (GH) secretion is markedly sexually dimorphic in rodents as in primates, but the neuroanatomical mechanisms of this phenomenon are not clear. In the arcuate nucleus of the hypothalamus, GH-releasing hormone (GHRH) neurones receive somatostatinergic inputs through the sst2A receptor (sst2A-R) and the percentage of GHRH neurones bearing sst2A-R is higher in female than in male GHRH-enhanced green fluorescent protein (eGFP) mice. In the present study, we hypothesised that sst2A-R expression on GHRH neurones is modulated by gonadal steroids and constitutes a mechanism for sexually differentiated GH secretion. The distribution of sst2A-R on GHRH neurones was evaluated by immunohistochemistry in adult GHRH-eGFP mice gonadectomised and treated for 3 weeks with oestradiol or testosterone implants. In gonadectomised females supplemented with testosterone, sst2A-R distribution on GHRH neurones was reduced to the level seen in intact males, whereas oestradiol implants were ineffective. Conversely, orchidectomy induced a female 'sst2A phenotype', which was reversed by testosterone supplementation. Changes in the hepatic expression of GH-dependent genes for major urinary protein-3 and the prolactin receptor reflected the altered steroid influence on GH pulsatile secretion. In the ventromedial-arcuate region, GHRH and sst2-R, as well as GHRH and somatostatin expression as measured by the real-time polymerase chain reaction, were positively correlated in both sexes. By contrast, the positive correlation between ventromedial-arcuate GHRH and periventricular somatostatin expression in males was reversed to a negative one in females. Moreover, the positive correlation between periventricular somatostatin and ventromedial-arcuate sst2-R expressions in males was lost in females. These results suggest that, in the adult mouse, testosterone is a major modulator of sst2A distribution on GHRH neurones. This marked sex difference in sst2A-R distribution may constitute a key element in the genesis of the sexually differentiated pattern of GH secretion, possibly through testosterone-modulated changes in somatostatin inputs from hypophysiotrophic periventricular neurones.

Elodie Chaillou and Dominique Grouselle contributed equally to this work.; International audience; As in other species, exogenous administration of ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptors can stimulates feeding behaviour and GH secretion in the sheep. However, the importance of endogenous ghrelin for these two functions as well as its central or peripheral origin remained to be established. In the present study, cerebrospinal fluid (CSF) ghrelin concentrations were measured in five anoestrous ewes and found to be more than 1000-fold lower than circulating plasma levels, in keeping with the even lower concentration in hypothalamic compared to abomasum tissue extracts. Cluster analysis indicated that CSF ghrelin levels were markedly pulsatile, with a greater number of peaks than plasma ghrelin. Pulsatility parameters were closer for GH and CSF ghrelin than between GH and plasma ghrelin. Plasma ghrelin and GH levels were significantly correlated in three out of five ewes but CSF ghrelin and GH in one ewe only. Half of the CSF ghrelin episodes were preceded by a ghrelin peak in plasma with a 22-min delay. Cross-correlations between plasma GH and plasma or CSF ghrelin did not reach significance but a trend towards cross-correlation was observed from 20 to 0 min between plasma and CSF ghrelin. At 09.00 h, when food was returned to ewes, voluntary food intake did not elicit a consistent change in plasma or CSF ghrelin levels. By contrast, a peripheral ghrelin injection (1 mg, i.v.) immediately stimulated feeding behaviour and GH secretion. These effects were concomitant with a more than ten-fold increase in plasma ghrelin levels, whereas CSF ghrelin values only doubled 40-50 min after the injection. This suggests that peripherally-injected ghrelin crosses the blood-brain barrier, but only in low amount and with relatively slow kinetics compared to its effects on GH release and food intake. Taken together, the results obtained in the present study support the notion that, in the ovariectomised-oestradiol implanted sheep model, peripheral ghrelin injection rapidly induces GH secretion, and feeding behaviour, probably by acting on growth hormone secretagogue receptor subtype 1 located in brain regions in which the blood-brain barrier is not complete (e.g. the arcuate nucleus).

International audience; Findings from animal studies have suggested that bone remodeling is under beta-adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrbeta2R and the absence of Adrbeta1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (-52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Young's Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrbeta2R-deficient mice, suggest that low doses of beta-blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues.

Phytoestrogens, naturally occurring plant compounds having oestrogenic and/or anti-oestrogenic activity, are present in many human foodstuffs including hop. Moderate intakes of isoflavonoid phytoestrogens have been associated with a reduction in cardiovascular diseases incidence. So, it is possible that hop ( Humulus Lupulus L.) might similarly contribute to the reported health-beneficial effects of moderate beer consumption. Thus, the purpose of this study was to investigate in vitro effects of aqueous hop extract on thoracic vascular reactivity in Sprague Dawley male and female rats. Endothelium-intact thoracic arterial rings from male rats (MALE, n =8), sham-ovariectomized (Sham OVX) female ( n =8) and ovariectomized (OVX) female rats ( n =8) were used. We assessed the relaxation induced by aqueous hop extract (10 −9 , 10 −2 g/l) in aortic rings precontracted with norepinephrine (10 −7 M), in the absence or in the presence of l -NAME (10 −4 M), indomethacin (10 −5 M), thapsigargin (10 −4 M), iberiotoxin (3.10 −8 M), apamin (3.10 −8 M) and TEA (3.10 −4 M). Aqueous hop extract induced relaxation of endothelium-intact thoracic arterial rings in MALE and Sham OVX rats, whereas a weak effect was observed in OVX rats. This vasorelaxation was strongly inhibited in presence of l -NAME, indomethacin and thapsigargin. These data indicated that aqueous hop extract-induced vasodilation, in male and intact female rats, is mediated by NOS activation, cyclooxygenase products and Ca 2+ pathways. Moreover, our results suggested that effect of hop in enhancing vascular reactivity was independent of gender but strongly related to hormonal status.

International audience; The ultradian pulsatile pattern of growth hormone (GH) secretion is markedly sexually dimorphic in rodents as in primates, but the neuroanatomical mechanisms of this phenomenon are not clear. In the arcuate nucleus of the hypothalamus, GH-releasing hormone (GHRH) neurones receive somatostatinergic inputs through the sst2A receptor (sst2A-R) and the percentage of GHRH neurones bearing sst2A-R is higher in female than in male GHRH-enhanced green fluorescent protein (eGFP) mice. In the present study, we hypothesised that sst2A-R expression on GHRH neurones is modulated by gonadal steroids and constitutes a mechanism for sexually differentiated GH secretion. The distribution of sst2A-R on GHRH neurones was evaluated by immunohistochemistry in adult GHRH-eGFP mice gonadectomised and treated for 3 weeks with oestradiol or testosterone implants. In gonadectomised females supplemented with testosterone, sst2A-R distribution on GHRH neurones was reduced to the level seen in intact males, whereas oestradiol implants were ineffective. Conversely, orchidectomy induced a female 'sst2A phenotype', which was reversed by testosterone supplementation. Changes in the hepatic expression of GH-dependent genes for major urinary protein-3 and the prolactin receptor reflected the altered steroid influence on GH pulsatile secretion. In the ventromedial-arcuate region, GHRH and sst2-R, as well as GHRH and somatostatin expression as measured by the real-time polymerase chain reaction, were positively correlated in both sexes. By contrast, the positive correlation between ventromedial-arcuate GHRH and periventricular somatostatin expression in males was reversed to a negative one in females. Moreover, the positive correlation between periventricular somatostatin and ventromedial-arcuate sst2-R expressions in males was lost in females. These results suggest that, in the adult mouse, testosterone is a major modulator of sst2A distribution on GHRH neurones. This marked sex difference in sst2A-R distribution may constitute a key element in the genesis of the sexually differentiated pattern of GH secretion, possibly through testosterone-modulated changes in somatostatin inputs from hypophysiotrophic periventricular neurones.

Estrogen plays a critical role in inducing LH surge. In the pituitary, estrogen receptor alpha (ERalpha) mediates the action of estrogen, while the downstream pathway of ERalpha activation is yet to be elucidated. Here, we report the finding that cholecystokinin type A receptor (CCK-AR) is an ERalpha downstream gene in the mouse anterior pituitary. In the cycling mouse pituitary, the expression of CCK-AR mRNA is markedly higher in the afternoon of proestrus compared with metestrus. Both ovariectomy (OVX) and null mutation of the ERalpha gene completely abolish CCK-AR mRNA expression. Injection of 17beta-estradiol to OVX wild-type mice induces recovery of CCK-AR mRNA expression to levels observed at proestrus, but no such recovery is induced in OVX ERalpha knockout mice. The same pattern of estrogen dependency in inducing CCK-AR mRNA expression was seen in cultured primary anterior pituitary cells, indicating that estrogen directly acts on pituitary cells to induce CCK-AR expression. Immunohistological analysis revealed that more than 80% of gonadotrophs express CCK-AR in the afternoon of proestrus. To test whether CCK-AR mediated the sensitizing effect of estrogen in GnRH-induced LH secretion, primary pituitary cells were primed with estrogen followed by treatment with GnRH in the presence or absence of lorglumide, a CCK-AR antagonist. While both groups secreted LH upon GnRH treatment, lorglumide treatment significantly decreased LH secretion. Taken together, this study finds CCK-AR to be an ERalpha downstream gene in the pituitary and suggests that CCK-AR may play a role in the estrogen sensitization of the pituitary response to GnRH.

Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.

International audience; The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.

Learning motor skills is critical for motor abilities such as driving a car or playing piano. The speed at which we learn those skills is subject to many factors. Yet, it is not known to what extent gonadal hormones can affect the achievement of accurate movements in time and space. Here we demonstrate via different lines of evidence that estradiol promotes plasticity in the cerebellar cortex underlying motor learning. First, we show that estradiol enhances induction of long-term potentiation at the parallel fiber to Purkinje cell synapse, whereas it does not affect long-term depression; second, we show that estradiol activation of estrogen receptor β receptors in Purkinje cells significantly improves gain-decrease adaptation of the vestibulo-ocular reflex, whereas it does not affect general eye movement performance; and third, we show that estradiol increases the density of parallel fiber to Purkinje cell synapses, whereas it does not affect the density of climbing fiber synapses. We conclude that estradiol can improve motor skills by potentiating cerebellar plasticity and synapse formation. These processes may be advantageous during periods of high estradiol levels of the estrous cycle or pregnancy.

Using female 4-week-old Sprague–Dawley rats, we investigated the effects of 14 weeks of progressive strength isometric training on endothelium dysfunction after estrogen deficiency. We also proposed possible mechanism(s) by which such training acted on endothelium-dependent vasodilation in thoracic aortic rings. Rats were randomly divided into 4 groups of 8 rats: a sham operated group, an ovariectomized sedentary group receiving 17β-estradiol vehicle s.c. daily, an ovariectomized sedentary group receiving a daily injection of 20 µg·kg–1 17β-estradiol s.c., and an ovariectomized exercised group receiving daily s.c. vehicle. Vascular reactivity of aortic rings have been evaluated by a cumulative dose of acetylcholine (ACh), in the presence or absence of l-NAME (N-nitro-l-arginine methyl ester), indomethacin, thapsigargin, iberiotoxin, apamin, and tetraethylammonium. Ovariectomy markedly decreased the relaxation caused by ACh, whereas 17β-estradiol treatment induced a significant increase in the relaxation elicited by ACh. Isometric exercise enhanced relaxation due to ACh. This enhancement was attenuated in the presence of l-NAME, indomethacin, thapsigargin, iberiotoxin, and apamin. Our data indicated, for the first time, that the endothelium-dependent relaxant response to ACh was markedly improved in trained ovariectomized rats. This increased vasodilation is mediated by nitric oxide, cyclooxygenase, sarco-endoplasmic reticulum Ca2+-ATPase pathways, and endothelium-derived hyperpolarizing factor. Finally, this study suggested that resistance training may provide benefits in addressing vascular dysfunction consequent to a decline in estrogen levels after menopause. However, any benefits for age-related vascular dysfunction remain to be demonstrated.

The active metabolite of vitamin A, retinoic acid (RA), plays an important role in the female reproductive system. The synthesis of RA is tightly regulated by the activity of retinaldehyde dehydrogenases (Raldh). Among these, Raldh1 and Raldh2 exhibit specific temporal and spatial expression patterns in the mouse uterus, both during the oestrous cycle and early pregnancy. In the present study, we have assessed whether oestradiol and progesterone directly influence the uterine expression of Raldh1 and Raldh2 in ovariectomised mice. We investigated the effect of gestagen (promegestone 0.3 mg kg−1 bodyweight), oestrogen (oestradiol 3 µg kg−1 bodyweight) and their combination on the uterine expression of Raldh2. Expression was analysed using in situ hybridisation and quantified using real-time detection reverse transcription–polymerase chain reaction. The results show that the expression of Raldh2 is rapidly (within 1–4 h) induced in stromal cells by oestrogen, but not by gestagen, treatment, whereas combined oestrogen + gestagen treatment leads to a more prolonged (48 h) response. In contrast, oestrogen, but not progesterone, treatment downregulates (within 4–24 h) Raldh1 expression in the uterine glandular epithelium. We conclude that the uterine RA concentrations are regulated by oestrogens via an effect on the expression of the Raldh synthesising enzymes. Such a regulation is consistent with the natural fluctuations of Raldh expression during the oestrous cycle, early pregnancy and blastocyst implantation.

ER alpha acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ER alpha signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. INTRODUCTION: Estrogen receptor alpha (ER alpha) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ER alpha knock-in [NERKI]) with heterozygote ER alpha knockout mice and studied the resulting female ER alpha(+/+), ER alpha(+/NERKI), and ER alpha(-/NERKI) mice. The only ER alpha present in ER alpha(-/NERKI) mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ER alpha(+/NERKI) mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. MATERIALS AND METHODS: BMD was measured using DXA and pQCT at 3 months of age (n = 46-48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 microg/day), or ovariectomy + estradiol (1.0 microg/day; n = 10-12/group) and restudied 60 days later. RESULTS AND CONCLUSIONS: At 3 months of age, both the ER alpha(+/NERKI) and ER alpha(-/NERKI) mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ER alpha(-/NERKI) mice were the opposite of those in ER alpha(+/+) mice. Relative to sham mice, ovariectomized ER alpha(-/NERKI) mice gained more bone (not less, as in ER alpha(+/+) mice), and estrogen suppressed this increase (whereas augmenting it in ER alpha(+/+) mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ER alpha(-/NERKI) versus ER alpha(+/+) mice. Collectively, these data show that alteration of the balance between classical and nonclassical ER alpha signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ER alpha signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.

International audience; In numerous fish species, dopamine has been found to strongly inhibit gonadotropin release. Among the enzymes that regulate dopamine turnover, tyrosine hydroxylase (TH), the rate-limiting anabolic enzyme, could be a target for endocrine feedback regulation. Since dopamine turnover is stimulated by estradiol in rainbow trout, we have investigated the effect of estradiol on TH and estradiol receptor expression. In situ hybridization was used to quantify mRNA levels in the brain of ovariectomized female rainbow trout implanted or not with estradiol pellets. We demonstrated that preoptic TH and estradiol receptor mRNA levels are greatly decreased by gonadectomy during vitellogenesis. For TH expression, this effect was reversed in part by estradiol supplementation. We have also confirmed the existence of an inhibitory gonadal feedback on FSH secretion, mediated by estradiol. The stimulating effect of estradiol on TH expression found in this study could be a pathway involved in gonadal feedback on gonadotropin release.

International audience; The aim of this study was to investigate the development of pulsatile GnRH secretion by GnRH neurones in primary cultures of olfactory placodes from ovine embryos. Culture medium was collected every 10 min for 8 h to detect pulsatile secretion. In the first experiment, pulsatile secretion was studied in two different sets of cultures after 17 and 24 days in vitro. In the second experiment, a set of cultures was tested after 10, 17 and 24 days in vitro to investigate the development of pulsatile GnRH secretion in each individual culture. This study demonstrated that (i) primary cultures of GnRH neurones from olfactory explants secreted GnRH in a pulsatile manner and that the frequency and mean interpulse duration were similar to those reported in castrated ewes, and (ii) pulsatile secretion was not present at the beginning of the culture but was observed between 17 and 24 days in vitro, indicating the maturation of individual neurones and the development of their synchronization.