Effect Of Exogenous Estrogen Research Articles

Primate models in women's health: inflammation and atherogenesis in female cynomolgus macaques (Macaca fascicularis)

Female cynomolgus monkeys are excellent models for understanding cardiovascular disease and the relationships between inflammatory processes and conditions such as atherogenesis. This review summarizes published research findings obtained through comprehensive, multidisciplinary, multi-investigator studies in nonhuman primates over the past two decades. These studies examined the effects of exogenous estrogens and dietary soy protein/isoflavones (IFs) on atherosclerosis, circulating biomarkers, and tissue inflammation in pre- and postmenopausal female cynomolgus monkeys. Inflammation may play a role in the initiation and progression of disease, be a consequence of the disease, or both. Circulating and tissue biomarkers with inflammatory and anti-inflammatory characteristics (including adhesion molecules such as e-selectin, VCAM-1, and ICAM-1, chemokines such as MCP-1, cytokines such as interleukins, and acute phase reactants such as CRP, and others) may be useful indicators of disease status. Treatment of postmenopausal subjects with estrogen resulted in significant reductions in several key inflammatory mediators as well as atherosclerosis, while dietary IF had a more limited effect on inflammation and atherogenesis. Circulating concentrations of key inflammatory proteins, including monocyte-chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6), were associated with atherosclerosis and lesion characteristics in these animals. In premenopausal female monkeys, a diet enriched in soy protein reduced arterial inflammation as well as atherogenesis in comparison to a diet enriched in casein-lactalbumin. Expression levels of arterial inflammation associated genes (MCP-1, ICAM-1) and markers for inflammatory cell types (macrophages and T cells) correlated with plaque size, were differentially influenced by treatments, and represent potential targets for interventions. Arterial expression of estrogen receptor alpha, the key mediator of estrogenic effects, was inversely correlated with plaque size and indices of inflammation, suggestive of an atheroprotective role. The findings provide additional evidence that circulating inflammatory markers (particularly MCP-1) may be useful indicators of atherosclerotic disease progression and responses to treatment in female primates, and that estrogens and dietary soy may inhibit atherogenesis in part through anti-inflammatory mechanisms.

Effects of the route of estrogen administration on insulinlike growth factor-I, IGF binding protein-3, and insulin resistance in healthy postmenopausal women

Oral estrogen therapy suppresses insulin like growth factor I (IGF-I) levels, whereas conventional dose transdermal estradiol (E2) does not. However, it has been proposed that if sufficiently high serum E2 levels are achieved, nonoral E2 will also suppress serum IGF-I. The aim of the study was to investigate the effects of intranasal E2 with norethisterone (E2/NET) versus oral E2/NET acetate on IGF-I, IGF binding protein 3, and insulin resistance in postmenopausal women. This was a randomized, multicenter, double-blind, double-dummy trial. Postmenopausal women were randomized to receive either daily intranasal E2/NET (175 microg/275 microg) as a spray and a placebo tablet (n = 41) or oral E2/NET acetate (1 mg/0.5 mg) plus placebo intranasal spray (n = 41) for 1 year. Fasting plasma concentrations of IGF-I, IGF binding protein 3, glucose and insulin, glucose and insulin at 120 minutes post-glucose challenge, and the homeostasis model assessment for insulin resistance were assessed at baseline and after 52 weeks of treatment. The two groups were well matched for all clinical and biochemical parameters at baseline. There were no significant between-group differences for fasting and 120-minute glucose, insulin, homeostasis model assessment for insulin resistance, and IGF binding protein 3. The mean IGF-I level at week 52 was significantly lower for women treated with oral versus intranasal therapy (116 +/- 21 [SD] versus 134 +/- 33 [SD], P = 0.005) and the mean difference in change over 52 weeks in IGF-I was significantly different between groups (-19, 95% CI:-37 to -1, P = 0.04). In healthy postmenopausal women, intranasal E2 at a dose that results in serum levels that exceed the proposed threshold for growth hormone and IGF-I effects, does not alter IGF-I levels. This suggests that the effect of exogenous estrogen on IGF-I is a function of the method of administration rather than being dose related.

Liver Dysfunction in Turner Syndrome: Prevalence, Natural History and Effect of Exogenous Estrogen

Women with Turner syndrome (TS), one of the commonest sex chromosomal abnormalities, are at increased risk of impaired liver function as manifested by elevated liver enzymes, but its clinical significance remains uncertain. Factors contributing to increased liver function tests (LFTs) were investigated in 3 studies: A cross-sectional review of liver function in 125 women with TS whose median age was 31 years. A longitudinal study of 30 women with TS who were followed-up for 8 years on average. A dose-response study of 14 women with TS and 11 control women with hypogonadism who received 17-β-estradiol in doses of 1, 2, and 4 mg daily cyclically for 12 weeks each. The enzymes measured included γ-glutamyl transferase (GGT), alanine aminotransferase (ALT), and alkaline phosphatase. Compared with a control population, 91% of women with TS had elevated liver enzymes. The annual incidence was 2.1%. In the cross-sectional study, the enzyme most frequently elevated was GGT. LFTs correlated positively with cholesterol levels and body mass index. Increasing doses of hormone replacement therapy were associated with significant reductions in GGT, ALT, bilirubin, and albumin. Exogenous estrogen, whether in the form of oral contraception or HRT, improved liver function. There was no evidence of hepatic autoimmunity apart from anti-smooth muscle antibodies in 2.2% of individuals. Previous studies using reference ranges rather than matched control subjects may have underestimated the frequency of elevated liver enzymes in women with TS. The investigators believe that hepatic dysfunction in women with TS is probably a form of hepatic steatosis. Estrogen therapy should continue despite the presence of liver dysfunction, and there may be reason to increase the estrogen replacement dose.

Effect of sex steroids on plasma C-type natriuretic peptide forms: stimulation by oestradiol in lambs and adult sheep

Although C-type natriuretic peptide (CNP) is crucial to post-natal endochondral growth, roles for the hormone in pubertal bone growth and the physiological effects of sex steroid substitution on CNP synthesis are not known. Using a plasma marker of CNP tissue synthesis (amino-terminal proCNP, NTproCNP), we have studied the effect of exogenous oestrogen (E(2)) or testosterone (T) on plasma CNP forms and bone alkaline phosphatase (bALP) in pre-pubertal lambs. Responses to E(2) in non-cycling adult ewes were also studied. In 15-week-old intact ewe lambs, E(2) promptly increased plasma NTproCNP and CNP (P<0.001) to peak on day 2, and bALP (P<0.001 peaking on day 7), whereas no significant stimulation in response to T was observed in male lambs. Linear bone growth and live weight were unaffected. In adult anoestrous ewes, basal concentrations of CNP forms and bALP were lower than in ewe lambs, in keeping with skeletal maturity, but adults responded similarly to E(2). Prompt and sustained increases in NTproCNP and CNP, and a later threefold rise in bALP (all P<0.001), were induced by E(2). Possible contributions to these increases in plasma CNP forms by reproductive tissues (a known site of E(2)-induced CNP expression) were excluded by showing similar E(2)-induced CNP responses in adult ewes after surgical removal of reproductive tissues. These results are the first to show that E(2) stimulates plasma CNP forms and bALP in lambs and adult sheep and raise the possibility that CNP also participates in bone formation in the mature skeleton.

Liver dysfunction in Turner's syndrome: prevalence, natural history and effect of exogenous oestrogen

Raised liver enzymes are a common feature of Turner's syndrome (TS), but the cause remains unclear. We studied the hepatic function in a large cohort of women with TS and tested the effect of increasing doses of hormone replacement therapy (HRT) on liver function tests (LFTs). LFTs were assessed in three studies. A cross-sectional review of liver function of 125 women (median age: 31 years), a longitudinal study of 30 women (mean follow-up period: 8 years) and a dose-response study of 14 women with TS and 11 controls with hypogonadism, who received oral 17-beta-oestradiol (E(2)) 1, 2 and 4 mg daily in a cyclical formulation for 12 weeks each. Clinical features, oestrogen use and metabolic parameters were compared to liver enzymes (gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)), albumin and bilirubin. LFTs were also measured during each treatment interval of the dose-response study. Hepatic autoimmunity was sought in the cross-sectional study. When compared to the control population, as opposed to reference ranges, 91% of women with TS demonstrated liver enzyme elevation, with a yearly incidence of 2.1%. LFTs correlated positively with cholesterol (P < 0.001), BMI (P = 0.004) and type of oestrogen therapy (P = 0.04). Increasing doses of HRT resulted in a significant decrease in GGT, ALT, bilirubin and albumin. No evidence of excessive hepatic autoimmunity was found. The prevalence of raised liver enzymes in TS may have been underestimated by the use of reference ranges rather than matched controls. Obesity and hyperlipidaemia are associated with raised LFTs, as well as the use of HRT compared to the oral contraceptive pill (OCP). Exogenous oestrogen both as OCP and HRT improves liver function. Liver dysfunction in TS is likely to be a form of hepatic steatosis and intervention trials are now indicated.

Obstetrics in elephants

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

Sexual Differentiation of the LH Surge Response to Estradiol in Sheep Treated Prenatally with Testosterone Plus an Antiandrogen.

The steroid feedback mechanisms regulating GnRH secretion are sexually differentiated by testicular steroids during prenatal development. In sheep, prenatal testosterone (T) treatment of developing females results in disruption of the GnRH surge response to estradiol (E) positive feedback. Defeminization of the surge mechanism is considered to be defeminized by the estrogenic metabolites of testosterone because the surge mechanism is functional in females treated prenatally with the non-aromatizable androgen, dihydrotestosterone (DHT). However, it is difficult to confirm this premise by studying the organizing actions of prenatal estrogens because of the disruptive effects of exogenous estrogens on the maternal uterus. We developed an approach to study the consequences of excess estrogens in the developing female by co-administering T and the antiandrogen flutamide, and used this approach to test the hypothesis that prenatal estrogens defeminize the GnRH surge response to E. We predicted that the surge would be defeminized by prenatal T and T+flutamide treatments, but not by prenatal DHT. Lambs born to untreated females or females treated with T, DHT, or T+flutamide from Days 30-90 of gestation were neonatally gonadectomized, and steroid levels were standardized with a chronic E implant (30-mm sc). At 8 months of age, the chronic implants were removed and females and T+flutamide males were treated with new implants to maintain luteal phase concentrations of progesterone (P) and E for 9 days. After 9 days, P implants were removed and additional E implants (4 x 30-mm) were inserted to maintain follicular phase concentrations of E. Plasma concentrations of LH were measured in venous blood samples collected every 2 hours from -4 to 72 h from E administration. An LH surge was produced in all untreated, control (C, n=6) and DHT-treated females (n=7), 1 of 9 T-treated females, 4 of 6 T+flutamide-treated females, and 3 of 7 T+flutamide males. The onset of the surge relative to E administration did not differ between C (18.7 ± 1.6 h) and DHT (16.9 ± 0.9 h) females, but it was delayed (p < 0.02) in the T+flutamide groups (females: 40.5 ± 6.9 h; males: 66.7 ± 1.8 h). The peak amplitude of the LH surge was not different between C (175.9 ± 13.7 ng/ml) and DHT (209.6 ± 18.0 ng/ml) females, but was lower in T+flutamide females (84.2 ± 15.3 ng/ml) and males (48.0 ± 14.9 ng/ml). The one LH surge produced by a T-treated female had an onset of 44 h and a peak amplitude of 30.3 ng/ml. The LH surge response in some of the T+flutamide females and males contradicts our prediction, and fails to support our hypothesis that prenatal estrogens, made available to the developing fetus by the aromatization of T, defeminize the positive feedback actions of estradiol. However, the LH surges in the T+flutamide animals were abnormal, particularly in the males. This suggests that endogenous androgens not blocked by flutamide treatment or having organizational actions outside of the treatment period contributed to differentiation of the surge mechanism. Alternatively, individual differences in neural aromatase activity could result in different levels of estradiol exposure during the treatment period. We conclude that prenatal T+flutamide treatment defeminizes the neural circuitry underlying the GnRH surge mechanism, but that both androgens and estrogens may both be necessary for complete ablation of the surge response. Supported by P01 HD-44232.

Postmenopausal vaginitis

The importance of vaginitis in postmenopausal women will increase as the US population ages. With the withdrawal of estrogen, the vaginal environment changes dramatically. Along with physical changes, an overall decrease in the normal Lactobacillus-dominant flora and Candida colonization occurs. Trichomoniasis-, vulvovaginal candidiasis, and bacterial vaginosis still occur in menopausal women but probably less commonly than in younger women. The effects of exogenous estrogen on these conditions remain unknown. Less common conditions such as desquamative inflammatory vaginitis and lichen sclerosus gain relative importance in this population.

Developmental Changes in Testicular Sensitivity to Estrogens throughout Fetal and Neonatal Life

There is now compelling evidence that inappropriate exposure to estrogen during fetal or neonatal life could affect adult reproductive functions because the testis is sensitive to estrogens during specific periods of its development. Therefore, we investigated the effects of exogenous estrogens on gametogenesis and steroidogenesis during fetal and neonatal testicular development in the rat. We used in vitro systems, organ cultures, and dispersed testicular cell cultures, which allow the development of fetal and neonatal germ cells (gonocytes) and Leydig cells. Exogenous estrogens inhibited testosterone production in dispersed testicular cell cultures throughout fetal life, but this inhibition was observed only in the early fetal stages in organ culture. By using an aromatase inhibitor (letrozole, Novartis Pharma AG), we showed that the inhibitory effect of exogenous estrogens on testosterone production is masked in the whole testis at later stages (20.5 days postconception) due essentially to local production of estrogens. In both systems, additions of high concentrations (10(-6) M) of 17beta-estradiol or diethylstilbestrol decreased the number of gonocytes during the first fetal proliferative period but not during the neonatal period. Letrozole was without effect, suggesting that the aging-related loss of responsiveness of gonocytes is not due to any aromatase activity in the gonocytes.

Estrogens Induced Male Production at a Female-Producing Temperature in a Reptile (Leopard Gecko, Eublepharis Macularius) with Temperature-dependent Sex Determination

Estrogens and estrogen mimics can affect offspring sex ratios in a wide variety of animal species including Leopard Geckos, Eublepharis macularius, a species with temperature-dependent sex determination (TSD). Estrogens can disrupt effects of temperature on sex determination by feminizing or sex-reversing embryos incubated at male-producing temperatures. Estrogens may have paradoxical effects at different incubation temperatures and in different concentrations. In this study, E. macularius eggs were exposed to ethanol, estradiol benzoate, or estradiol 17b at the beginning of the thermosensitive period when sex determination occurs. Eggs were treated and incubated at each of three incubation temperatures known to produce varying primary sex ratios. At a male-producing incubation temperature, estrogen-treated groups produced more females than negative control groups. This result has been reported in other TSD reptiles. In our study, we showed that at a female-producing incubation temperature, estrogen-treated groups produced significantly more males than negative control groups. This is the first report of its kind in which a TSD reptile was shown to produce significantly more males at a female-producing temperature. Our results suggest a complex feedback relationship between aromatase and cofactors during the thermosensitive period of egg incubation in E. macularius. Expected effects of exogenous estrogens on contaminant-exposed wildlife populations may differ depending on nest-site temperatures. In species with temperature-dependent sex determination (TSD), sex is determined by the temperature to which an embryo is exposed during a thermally sensitive period of incuba- tion. The influence of exogenous estrogens on

SirT1 modulates the estrogen–insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice

IntroductionEstrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-κB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice.MethodsSirT1-deficient (SirT1ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region.ResultsBoth male and female SirT1ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1ko/ko mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1ko/ko mammary tissues, but not that of IκBα expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFα treatment enhanced the level of the newly synthesized IκBα in SirT1ko/ko cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals.ConclusionSirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells.

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.

Osteocytes Use Estrogen Receptor α to Respond to Strain but Their ERα Content Is Regulated by Estrogen

The role of mechanical strain and estrogen status in regulating ERalpha levels in bone cells was studied in female rats. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas habitual strain and artificial loading has only a small but positive effect, except on the ulna's medial surface, where artificial loading stimulates reversal of resorption to formation. Osteoporosis is the most widespread failure of bones' ability to match their architectural strength to their habitual load bearing. In men and women, the severity of bone loss is associated with bioavailability of estrogen. This association could result from the estrogen receptor (ER) involvement in bone cells' adaptive response to loading. In vivo semiquantitative analysis of the amount of ERalpha protein per osteocyte was performed in immuno-cytochemically stained sections from control and loaded rat ulna, as well as tibias of ovariectomy (OVX) and sham-operated female rats. In vitro, the effect of exogenous estrogen (10(-8) M) and mechanical strain (3400 microepsilon, 1 Hz, 600 cycles) on the expression of ERalpha mRNA levels was assessed in ROS 17/2.8 cells in monolayers using real-time PCR and ER promoter activity. ERalpha translocation in response to exogenous estrogen and mechanical strain was assessed in both ROS 17/2.8 and MLO-Y4 cells. More than 90 percent of tibial osteocytes express ERalpha, the level/osteocyte being higher in cortical than cancellous bone. OVX is associated with decreased ERalpha protein expression/osteocyte, whereas in the ulna habitual strain and that caused by artificial loading had only a small but positive effect, except on the medial surface, where loading stimulates reversal of resorption to formation. In unstimulated osteocytes and osteoblasts in situ, and osteocyte-like and osteoblast-like cells in vitro, ERalpha is predominantly cytoplasmic. In vitro, both strain and estrogen stimulate transient ERalpha translocation to the nucleus and transient changes in ERalpha mRNA. Strain but not estrogen also induces discrete membrane localization of ERalpha. Bone cells' responses to both strain and estrogen involve ERalpha, but only estrogen regulates its cellular concentration. This is consistent with the hypothesis that bone loss associated with estrogen deficiency is a consequence of reduction in ERalpha number/activity associated with lower estrogen concentration reducing the effectiveness of bone cells' anabolic response to strain.

S1-01-04: Estrogens / endogeneous & exogeneous

Cognitive impairment affects at least 10% of elders aged 65 years and older, increasing in prevalence to 50% of elders 85 years of age and older. In hopes of identifying agents that may prevent cognitive impairment, there has been tremendous interest in the role of both exogenous and endogenous hormones and risk of Alzheimer's disease (AD) and pre–clinical cognitive decline, especially in women. Indeed, estogen receptors (ER) (both ER α and ER β) are located throughout the brain, especially in regions involved in learning and memory such as the hippocampus and amygdala. However, studies in women involving estrogens have been conflicting with the predonderance of observational studies suggesting benefit and the randomized trials suggesting no benefit and possible harm. We will review the current evidence of the effect of exogenous estrogen and the risk of developing dementia. In addition, we will discuss results from studies of selective estrogen receptor modulators (SERMs) and highlight areas of future research with these and other novel estrogenic compounds. We will also review the literature on endogenous estrogens and cognitive function and risk of AD and suggest future directions for research.

Hormonal treatments and epithelial ovarian cancer risk

Exogenous sex hormones are widely used by women either for pregnancy prevention, as part of infertility treatment, or for treatment of menopausal symptoms. The role of these hormones in the development of ovarian cancer has been vastly explored. The protective effect of combined oral contraceptive pill is confirmed in multiple studies, but it is not clear whether this protection also covers women with a genetic predisposition to ovarian cancer. There is no conclusive evidence of infertility treatments increasing ovarian cancer risk, but infertility as such is a risk factor. Currently available data suggest that long-term users of hormone replacement therapy may have a slightly increased risk for ovarian cancer compared to women who have never used estrogen. The risk might particularly involve the endometrioid type of ovarian cancer. Most data on ovarian cancer and estrogen comes from epidemiological studies, since the normally high concentrations of estrogens in ovarian tissue and follicular fluid make direct biologic studies on the effects of exogenous estrogens on the ovarian cell difficult. This review discusses the risk of ovarian cancer associated with the use of sex steroid hormones, with special emphasis on the possible risk associated with estrogens.

Exogenous Estrogen Boosts Circulating Estradiol Concentrations and Calcium Uptake by Duodenal Tissue in Heat-Stressed Hens

In the hen, heat stress (HS) disrupts shell calcification and reproductive processes, including hormone synthesis and egg production. Two studies were conducted to investigate palliative effects of exogenous estrogen or dietary vitamin D3 on Ca homeostasis and reproductive physiology during HS. Study 1: Hy-Line W36 hens were randomly assigned to thermoneutral (TN) or HS treatments and to 1 of 7 estrogen treatments: zero (control) or one Compudose 200 implant given 1, 2, 3, 8, 9, or 10 d before onset of HS. With no implant, HS reduced plasma estradiol (E2) and total Ca absorbed (CaT) by duodenal cells (P < 0.05). In TN hens with implants, plasma E2 tripled within 24 h (P < 0.05) and remained elevated (P < 0.05) through d 9. In HS hens with implants, plasma E2 rose 6-fold (P < 0.05) to equal TN+E2 concentrations and remained elevated through d 10. In TN and HS hens with implants, the rate of Ca absorption (CaTR) and CaT increased dramatically; the responses were quadratic and essentially identical. Study 2: Hy-Line W36 hens were provided diets formulated either according to NRC requirements (NRC, 1994), or with the addition of 22,000 IU/kg vitamin D3 (+VD hens). A 24-h HS episode was imposed 2 wk after initiation of the dietary regimen. Duodenal samples were collected for Ca absorption assays after the 24-h HS episode. Both CaTR and CaT in +VD hens were approximately 3-fold higher than in hens in the NVD group (P = 0.102). The results lead to the conclusion that exogenous estrogen, high levels of dietary vitamin D, or both, before a HS episode, are efficacious in alleviating at least some of the effects of HS and should be further investigated.

Differential effects of 17beta-estradiol upon stroke damage in stroke prone and normotensive rats.

We previously reported that during pro-estrus (high endogenous estrogen levels), brain damage after middle cerebral artery occlusion (MCAO) was reduced in stroke-prone spontaneously hypertensive rats (SHRSP) but not in normotensive Wistar Kyoto rat (WKY). In the present study, we examined the effect of exogenous estrogen on brain damage after MCAO in SHRSP and WKY. A 17beta-estradiol (0.025 mg or 0.25 mg, 21 day release) or matching placebo pellet was implanted into ovariectomized WKY and SHRSP (3 to 4 months old) who then underwent distal diathermy-induced MCAO 2 weeks later. Plasma 17beta-estradiol levels for placebo and 17beta-estradiol groups were as follows: WKY 0.025 mg 16.4 +/- 8.5 (pg/mL, mean +/- SD) and 25.85 +/- 12.6; WKY 0.25 mg 18.2 +/- 9.0 and 69.8 +/- 27.4; SHRSP 0.25 mg 20.7 +/- 8.8 and 81.0 +/- 16.9. In SHRSP, infarct volumes at 24 hours after MCAO were similar in placebo and 17beta-estradiol groups: SHRSP 0.025 mg 126.7 +/- 15.3 mm (n = 6) and 114.0 +/- 14.1 mm (n = 8) (not significant); SHRSP 0.25 mg 113.5 +/- 22.3 mm (n = 8) and 129.7 +/- 26.2 mm (n = 7) (not significant), respectively. In WKY, 17beta-estradiol significantly increased infarct volume by 65% with 0.025 mg dose [36.1 +/- 20.7 mm (n = 8) and 59.7 +/- 19.3 mm (n = 8) (P = 0.033, unpaired t-test)] and by 96% with 0.25 mg dose [55.9 +/- 36.4 mm (n = 8) and 109.7 +/- 6.7 mm (n = 4) (P = 0.017)]. Thus, 17beta-estradiol increased stroke damage in normotensive rats with no significant effect in stroke-prone rats. Despite being contrary to our hypothesis, our findings add substance to the recently reported negative effects of 17beta-estradiol in clinical studies.

Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application

We aimed to assess the effect of ovariectomy on cartilage turnover and degradation, to evaluate whether ovariectomized (OVX) rats could form an experimental model of postmenopausal osteoarthritis. The effect of ovariectomy on cartilage was studied using two cohorts of female Sprague–Dawley rats, aged 5 and 7 months. In a third cohort, the effect of exogenous estrogen and a selective estrogen receptor modulator was analyzed. Knee joints were assessed by histological analysis of the articular cartilage after 9 weeks. Cartilage turnover was measured in urine by an immunoassay specific for collagen type II degradation products (CTX-II), and bone resorption was quantified in serum using an assay for bone collagen type I fragments (CTX-I). Surface erosion in the cartilage of the knee was more severe in OVX rats than in sham-operated animals, particularly in the 7-month-old cohort (P = 0.008). Ovariectomy also significant increased CTX-I and CTX-II. Both the absolute levels of CTX-II and the relative changes from baseline seen at week 4 correlated strongly with the severity of cartilage surface erosion at termination (r = 0.74, P < 0.01). Both estrogen and the selective estrogen receptor modulator inhibited the ovariectomy-induced acceleration of cartilage and bone turnover and significantly suppressed cartilage degradation and erosion seen in vehicle-treated OVX rats. The study indicates that estrogen deficiency accelerates cartilage turnover and increases cartilage surface erosion. OVX rats provide a useful experimental model for the evaluation of the chondroprotective effects of estrogens and estrogen-like substances and the model may be an in vivo representation of osteoarthritis in postmenopausal women.

238ESTROGEN RECEPTOR ALPHA AND PROGESTERONE RECEPTOR EXPRESSION FROM REPRODUCTIVE TISSUE AND IN VITRO PRODUCED EMBRYOS OF THE DOMESTIC CAT

The in vitro production of cat embryos has been reported by several laboratories, and kittens have been born after transfer of embryos derived by IVF, ICSI, and NT. However, evidence accumulating in other species indicates that in vitro-derived embryos exhibit altered gene expression of developmentally important genes. Because the domestic cat genome is not well defined, the lack of primer sequence information poses a challenge for gene expression profiling. Estrogen, in addition to its essential role in the development and function of the female reproductive tract, is important for maturation of the oocyte. The aim of this preliminary study was to evaluate the expression profile of estrogen receptor alpha (ERα) and progesterone receptor (PR) in domestic cat female reproductive tissue and in vitro-produced (IVP) embryos. Embryos were produced in vitro as described by Gomez et al. (2003 Theriogenology 60, 239–251). mRNA was isolated from pools (n=35–50) of IVP domestic cat embryos at the morula and blastocyst stages (Days 7 and 8) using a modified protocol of the PolyAT Tract System (Promega, Madison, WI, USA). Uterus and ovaries were collected from hystorectomized cats and total RNA was isolated from these estrogen-targeted organs (RNeasy standard protocol, QIAGEN, Valencia, CA, USA). All RNA was reverse-transcribed and subjected to real-time PCR to evaluate expression of ERα and PR. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for expression in all tissues. Using primers designed to amplify human sequences or multispecies primers, we successfully amplified all three genes from as little as ∼100ng of ovarian or uterine mRNA. We were also able to detect ERα and GAPDH from pools of IVP embryos. Transcripts were cloned and confirmed by sequencing to be homologous to known sequences of the respective genes in a variety of species, including human, mouse, and pig. A 234-bp transcript of ERα (accession #AY349164;; GenBank) corresponding to exons 5 through 7 of the human ERα gene (hormone-binding domain) was identified along with a 110-bp sequence of PR and 98-bp sequence of GAPDH. To our knowledge, this is the first description of ERα or PR cloning in the domestic cat. In summary, we have demonstrated that highly sensitive real-time PCR is effective for the assessment of gene expression in cat ovarian and uterine tissue, as well as in embryos, the latter, of which, are known to have a low transcript copy number. Furthermore, these experiments provide the basis for future studies on the effect of exogenous estrogen on gene expression in cat IVM oocytes and IVP embryos.

THE EFFECT OF NEONATAL ADMINISTRATION OF SOME DRUGS ON THE ESTROGEN RECEPTORS IN THE PROSTATE OF ADULT ALBINO RAT: A HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY

Normal development and function of male reproductive organs are primarily controlled by androgens (N;u et al., 2003). However, estrogen plays also a role in normal development although it is not well defined but this refers to that estrogen receptors (ER) are also present throughout the male reproductive tract. Some studies have documented the effects of exogenous estrogen on reproductive organs of both developing (McLachlan, 1981) and mature (Junlewicz, et aI., 1989) males. Administration of estrogen to developing fetuses or neonates produces pronounced short - and long - term effects on the growth, histology, and functional activity of reproductive organs (William et aI., 1999).