Amounts Of Estradiol Research Articles

Effect of Estradiol on miR-21& miR-155 Expression in promyelocytic leukemia-derived cell line NB4

Background: Due to the estrogen participation in modulating the proliferation and commitment of stem cells and the effects of miR-21 and miR-155 expression on reduced proliferation and colony formation of acute myeloid leukemia (AML), the aim of the present study was to evaluate the effect of estradiol on expression of miR-21 and miR-155 in the NB4 cell line, as an acute promyelocytic leukemia (APL). Materials and Methods: In the present experiment, NB4 cells were treated with different quantities of estradiol (5, 25, 50, 75, 100, 150, 200, 250 μg/ml) and vehicle control for 24 and 48 hours. Viability, apoptosis, and cellular proliferation were estimated by trypan blue exclusion, flow cytometry, and MTT assays, respectively. The level of miR-155 and miR-21 expression was studied using absolute quantitative real-time PCR. Results: Results showed that estradiol in the effective dose (200 μg/ml) led to decreased cellular viability (in a dose dependent manner, P = 0.004) and apoptosis of NB4 cells. In addition, the expressions of miR-155 and miR-21 were significantly and dose-dependently decreased (p<0.05). Conclusion: Estradiol at the effective dose caused apoptosis in NB4 cell line. This substance can be used as a drug for the treatment of APL. However, further assessments are needed to support the effectiveness of estradiol in the treatment of APL.

Simultaneous Electrochemical Detection of Estradiol and Testosterone Using Nickel Ferrite Oxide Doped Mesoporous Carbon Nanocomposite Modified Sensor

Estradiol levels in women are related to lung, uterine (endometrial), ovarian and breast cancers, although the exact mechanism of cancer development is not fully understood. A new cost-effective sensor was fabricated by incorporating conductive NiFe2O4 metal oxide into the electrochemical sensitive platform of mesoporous carbon (MC) derived from cotton to measure estradiol determination by electrochemical methods. A new modified GCE/NiFe2O4-MC electrode, which exhibits electrocatalytic activities, was created by dropping NiFe2O4-MC onto the glassy carbon electrode (GCE) surface using the drop casting technique to detect the electroactive estradiol molecule. Estradiol was determined electrochemically by using the square wave voltammetry technique with a detection limit of 6.88 nM in 20.0–566 nM linear range in phosphate buffer solution at pH 7.4 with this novel modified GCE/NiFe2O4-MC electrode, that has the best electron transfer rate. The simultaneous determination of estradiol in the presence of testosterone was successfully performed in 0.1 M tetrabutylammonium tetrafluoroborate supportive electrolyte solution prepared in acetonitrile, an anhydrous medium. Real sample applications were carried out to prove the applicability and precision of the novelty-produced electrode. The amount of estradiol in tablet samples was obtained satisfactorily with high recovery values by standard addition method.

The Association Between Progesterone, Estradiol, and Oxytocin and Heat Pain Measures in Pregnancy: An Observational Cohort Study.

Hormonal action has been implicated as a possible mechanism for pregnancy-induced analgesia. Previous investigators have reported an increase in heat pain tolerance during labor compared with nonpregnant controls and postulated it was because of the hormonal changes during pregnancy. However, these previous reports did not include measurement of hormonal values. The purpose of our study was to quantitatively test if changes in pregnancy hormone concentrations correlated with changes in temperature ratings. This was a prospective cohort study consisting of 32 women scheduled for elective cesarean delivery at term between July 2010 and January 2013. Heat pain threshold and tolerance, estrogen, progesterone, and oxytocin levels were measured twice in each patient at term and again 4 to 8 weeks postpartum. All hormone levels decreased significantly between term pregnancy and the postpartum visit (all P values < 0.029). However, there were no statistically significant differences between term and postpartum heat pain measurements. The mean baseline heat pain threshold was 40.9°C at term compared with 40.3°C °postpartum (P = 0.47; mean change, -0.6°C; 95% confidence interval of change, -1.8°C to +0.7°C). The mean baseline heat pain tolerance was 46.1°C at term and 46.0°C postpartum (P = 0.59; mean change, -0.1°C; 95% confidence interval of change, -0.8°C° to +0.6°C). Our findings show that amounts of estradiol and progesterone changed significantly between the term and the postpartum visit; however, the thermal pain tolerance did not significantly change. In summary, we did not observe an association between hormonal changes and changes in pain threshold measures. This finding argues against the concept of simple progesterone- or estrogen-induced analgesia in humans.

A microsomal based method to detect aromatase activity in different brain regions of the rat using ultra performance liquid chromatography–mass spectrometry

Aromatase (ARO) is a cytochrome P450 enzyme that accounts for local estrogen production in the brain. The goal of this study was to develop a microsomal based assay to sensitively and reliably detect the low levels of ARO activity in different brain regions. Enzyme activity was detected based on the conversion of testosterone to estradiol. Quantity of estradiol was measured using ultra performance liquid chromatography–mass spectrometry. Detection was linear over a range of 2.5–200pg/ml estradiol, and was reproducible with intra- and inter-assay coefficients of variation (CV) <15%. Estradiol production using isolated microsomes was linear with time up to 30min as well as linearly related to amount of microsome. Substrate concentration curves revealed enzymatic kinetics (hippocampus: Vmax and Km: 0.57pmol estradiol/h per mg microsome and 48.58nM; amygdala: Vmax and Km: 1.69pmol estradiol/h per mg microsome and 48.4nM; preoptic area: Vmax and Km: 0.96pmol estradiol/h per mg microsome and 44.31nM) with testosterone used at a saturating concentration of 400nM. Anastrozole treatment blocked ARO activity in hippocampal and ovarian microsomes, indicating that the assay is specific for ARO. Also, we showed that the distribution of the long form ARO mRNA (CYP19A1) in different regions of the brain is correlated with ARO activity, with highest levels in the amygdala, followed by preoptic area and hippocampus. In the frontal cortex, very little long form ARO mRNA, and little to no ARO activity, were detected. These findings demonstrate that the microsomal incubation (MIB) assay is a sensitive and reliable method for quantifying ARO activity in discrete brain regions.

Application of fly ash as an adsorbent for Estradiol in animal waste

The contamination of agricultural ground with estrogen compounds through application of animal wastes is a present concern. At the same time, current uses for waste fly ash having high carbon content are limited. To help mitigate these problems, we examine using waste fly ash as a useful adsorbent for Estradiol in pig waste digests. In this study, Estradiol was added to vials containing water and fly ash from several different power plants. After an extraction process, the amount of Estradiol in the water was measured. Commercial activated carbon was also used for comparison purposes. Vials containing varying concentrations of Estradiol and no trapping material were used as a control. The results from this study indicate that fly ash can be used as a trapping material for Estradiol in water, but that commercially available activated carbon can trap about an order of magnitude more Estradiol than the fly ash and that the effects of the fly ash matrix can both inhibit and promote the solvation of Estradiol into water depending possibly upon pH and cation concentration effects. In addition, preliminary extraction studies using pig waste digest indicate that fly ash can be used as adsorbent for Estradiol present in pig waste.

Effect of SIRT1 on cellular apoptosis and steroidogenesis in bovine ovarian granulosa cells in vitro

Sirtuin type 1 (SIRT1) gene plays an important role in the regulation of female reproduction, and we set out to determine if SIRT1 affects steroidogenesis. In this study, granulosa cells isolated from bovine ovaries were cultured and treated with resveratrol or nicotinamide, a SIRT1 activator or inhibitor, respectively. The mRNA and protein levels of SIRT1 of cultured granulosa cells were investigated by quantitative real-time RT-PCR and Western blot, and then the extent of cellular apoptosis was measured by FACS assay. The amounts of estradiol and progesterone in cultured media were measured by radioimmunoassay, and mRNA and protein levels of Steroidogenic Factor 1 (SF-1) were also investigated. We demonstrated that resveratrol exhibited a dose-dependent induction of SIRT1 expression, associated with late apoptosis in granulosa cells, while nicotinamide decreased SIRT1 expression with no effect on apoptosis. We observed the estradiol levels and SF-1 expression decreased while progesterone level increased after the treatment of resveratrol, but the opposite results were observed in the treatment of nicotinamide. In conclusion, SIRT1 affected apoptosis, steroidogenesis and the related gene expression in granulosa cells, which may provide new insight into the treatment of follicular and/or luteal phase deficiency.

Tratamiento de la atrofia vaginal a través de anillo con liberación de ultrabaja dosis de estradiol

Vaginal atrophy is a chronic disease that is often undiagnosed and has a major impact on sexual health and quality of life. Vaginal atrophy is caused by physiological changes due to a decrease of estrogens. These changes are the basis of a number of symptoms including vaginal dryness, dyspareunia, itching and irritation reported in 40-60% of postmenopausal women in Europe. The percentage of treated women is less than 30%, and compliance is less than 10% per year. Local estrogens are the first-line treatment for moderate-to-severe vaginal atrophy. A new local estrogen treatment has recently become available in Spain, consisting of a flexible silicone ring with a core containing 2 mg of estradiol hemihydrate. The amount of estradiol released is minimal but in continuous quantities, with a release of 6.5 to 9.5 mcg/day for at least 90 days. The vaginal ring has been proven to be effective in relieving local symptoms (vaginal and urological), improving the vaginal mucosa based on visual examination (pale color, turgor, roughness, and thickness) and on the rate of cytologic maturation (a greater proportion of surface cells and a decrease in basal, parabasal and intermediate cells) and pH. The ring produces the same side effects as other types of local estrogen and is the most frequently chosen method by patients because of its ease of use and comfort, and the general satisfaction produced.

Comparing the growth and the development of mouse pre-antral follicle in medium with PL (Platelet Layset) and with FBS

Introduction: In recent years a great deal of effort has been made to improve the culture medium which can be used for different cell culturing and Folliculogenesis. PL (Platelet Layset) with a high percentage of growth factors and also microelements can be effective on the growth of follicle and oocyte. Material and method: Preantral follicles were collected from 12 to 14 days female NMRI mice and cultured in culture medium with a different supplement as the serum for 12 days. The different serums were, 5% and 10% FBS as the control groups and the experimental groups were enriched by 5% PL, 10% PL and a combination of 5% PL and 5% FBS. The growth and development of follicle and oocyte was monitored and also the amount of Estradiol (E2) and progesterone (P4) was detected in different mediums on days 9 and 12 of culturing. Result: Oocytes had a significant growth in all medium compared to day zero and reached to the size of a mature oocyte (p < 0.05). Although survival rate after one day had significant decrease in all medium compared to 5% FBS (p < 0.05), after 12 days of culturing, the best survival rate belonged to 5% PL. Progesterone secretion increased in experimental groups significantly (p < 0.05) while Estradiol secretion in 5% and 10% PL decreased in half of control groups. Discussion: This result demonstrates that PL can be a considerable supplement that can be added to the culture medium or a good replacement for FBS as serum used in Folliculogenesis.

Use of Chitosan Membranes to Mimic Human Epidermis for the Development of an Estradiol Delivery System

Polymeric membranes with skin-mimetic permeability properties appear as attractive models for use in the development of transdermal drug delivery systems. These membranes can be placed in contact with the transdermal system and simulate the skin barrier. In the current work, statistical experimental design was implemented for preparation of the optimized chitosan membrane to mimic human epidermis. For this purpose, an optimization process based on response surface methodology was carried out in order to determine the best combination of independent factors for membrane preparation. The independent factors analyzed were concentration of chitosan, concentration of cross-linking agent, and cross-linking time. The optimized membrane successfully simulated the cumulative amount of estradiol released per unit time for human epidermis. In addition, the optimized membrane was evaluated for the development of an estradiol delivery system. This in vitro technology may offer an alternative methodology for the development of transdermal delivery systems.

Effects of red clover extracts on breast cancer cell migration and invasion

Postmenopausal hormone therapy is associated with increased incidence of breast cancer. For this reason alternative therapeutic options to treat menopausal symptoms have been developed. Red clover extracts (RCE) are rich in isoflavones, particularly genistein and daidzein and they have been proved to be effective in reducing vasomotor symptoms in a number of studies. Due to their partial selectivity of action on estrogen receptors (ERs) these compounds have been claimed to be safer on the breast. In this article, we explored the action of RCE on motility and invasion of ER positive breast cancer cells and we partially characterized the signaling mechanisms. The principal isoflavones contained in RCE acted as weak estrogenic compounds when administered alone. However, when provided in association with physiological amounts of estradiol, RCE acted as estrogen antagonist on remodeling of actin cytoskeleton that are requested to enact cell movement and with related modifications of the activity of actin-binding proteins, such as moesin. These results offer novel information on the molecular actions of isoflavones contained in red clover on breast cancer cells, supporting a possible action of these molecules as natural selective estrogen receptor modulators in the presence of physiological amounts of estrogens.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 2: Effects on reproductive parameters, on sex behavior, on memory retention and on hypothalamic expression of aromatase and 5alpha-reductases in the offspring

The gender-specific expression pattern of aromatase and 5alpha-reductases (5alpha-R) during brain development provides neurons the right amount of estradiol and DHT to induce a dimorphic organization of the structure. Polychlorinated biphenyls (PCBs) are endocrine disruptive pollutants; exposure to PCBs through placental transfer and breast-feeding may adversely affect the organizational action of sex steroid, resulting in long-term alteration of reproductive neuroendocrinology. The study was aimed at: a) evaluating the hypothalamic expression of aromatase, 5alpha-R1 and 5alpha-R2 in fetuses (GD20), infant (PN12), weaning (PN21) and young adult (PN60) male and female rats exposed to PCBs during development; b) correlating these parameters with the time of testicular descent, puberty onset, estrous cyclicity and copulatory behavior; c) evaluating possible alterations of some non reproductive behaviors (locomotion, learning and memory, depression/anxiety behavior). A reconstituted mixture of four indicator congeners (PCB 126, 138, 153 and 180) was injected subcutaneously to dams at the dose of 10 mg/kg daily from GD15 to GD19 and then twice a week till weanling. The results indicated that developmental PCB exposure produced important changes in the dimorphic hypothalamic expression of both aromatase and the 5alpha-Rs, which were still evident in adult animals. We observed that female puberty onset occurs earlier than in control animals without cycle irregularity, while testicular descent in males was delayed. A slight but significant impairment of sexual behavior and an important alteration in memory retention were also noted specifically in males. We conclude that PCBs might affect the dimorphic neuroendocrine control of reproductive system and of other neurobiological processes.

Estrogen Receptors Alpha (ESR1) and Beta (ESR2) Are Expressed in Circulating Human Lymphocytes

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23–50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro

There has been controversy over the role of FSH in the regulation of preantral follicle development. LH is a survival and differentiation factor that increases oocyte maturation in FSH-supplemented cultures of mouse preantral follicles. However, little information exists on the action of LH and FSH in the developmental competence of porcine preantral follicle oocytes in vitro. Porcine preantral follicles were cultured for 3 days in the presence or absence of FSH or LH. Oocytes from these follicles were then matured, fertilized in vitro, and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. FSH or combined LH and FSH significantly enhanced follicular growth compared to LH alone or the controls. Combined LH and FSH treatment of preantral follicles significantly increased the percentage (59 +/- 5%) of oocytes competent to undergo cleavage to the two-cell stage after fertilization. A significant effect was seen on oocyte competence to develop from the two-cell to the blastocyst stage (30 +/- 6%) compared to FSH alone treatment (45 +/- 7 and 14 +/- 5%, respectively). The amount of estradiol on days 2 and 3 of culture was significantly higher in follicles cultured with FSH (48.75 +/- 17, 70.5 +/- 14 pg/ml) or combined LH and FSH (63.25 +/- 16, 72.5 +/- 12 pg/ml) than that cultured with the untreated controls (16 +/- 10, 5.66 +/- 4 pg/ml). The results indicated that FSH is essential for the in vitro growth of porcine preantral follicles, estradiol secretion, and for oocytes to acquire competence to resume meiosis and undergo fertilization and embryonic development. LH with FSH treatment of porcine preantral follicles can improve the quality of oocytes by promoting growth and a higher frequency of embryonic development.

Judge, jury and executioner: the auto-regulation of luteal function

Experiments were conducted to further our understanding of the cellular and molecular mechanisms that regulate luteal function in ewes. Inhibition of protein kinase A (PKA) reduced (P < 0.05) secretion of progesterone from both small and large steroidogenic luteal cells. In addition, the relative phosphorylation state of steriodogenic acute regulatory protein (StAR) was more than twice as high (P < 0.05) in large vs small luteal cells. Large steroidogenic luteal cells appear to contain constitutively active PKA and increased concentrations of phosphorylated StAR which play a role in the increased basal rate of secretion of progesterone. To determine if intraluteal secretion of prostaglandin (PG) F2alpha was required for luteolysis, ewes on day 10 of the estrous cycle received intraluteal implants of a biodegradable polymer containing 0, 1 or 10 mg of indomethacin, to prevent intraluteal synthesis of PGF2alpha. On day 18, luteal weights in ewes receiving 1 mg of indomethacin were greater (P < 0.05) than controls and those receiving 10 mg were greater (P < 0.05) than either of the other two groups. Concentrations of progesterone in serum were also increased (P < 0.05) from days 13 to 16 of the estrous cycle in ewes receiving 10 mg of indomethacin. Although not required for decreased production of progesterone at the end of the cycle, intraluteal secretion of PGF2alpha appears to be required for normal luteolysis. To ascertain if oxytocin mediates the indirect effects of PGF2alpha on small luteal cells, the effects of 0, 0.1, 1 or 10 mM oxytocin on intracellular concentrations of calcium were quantified. There was a dose-dependent increase (P < 0.05) in the number of small luteal cells responding to oxytocin. Thus, oxytocin induces increased calcium levels and perhaps apoptotic cell death in small luteal cells. Concentrations of progesterone, similar to those present in corpora lutea (approximately 30 microg/g), prevented the increased intracellular concentrations of calcium (P < 0.05) stimulated by oxytocin in small cells. In large luteal cells the response to progesterone was variable. There was no consistent effect of high quantities of estradiol, testosterone or cortisol in either cell type. It was concluded that normal luteal concentrations of progesterone prevent the oxytocin and perhaps the PGF2alpha-induced increase in the number of small and large luteal cells which respond to these hormones with increased intracellular concentrations of calcium. In summary, large ovine luteal cells produce high basal levels of progesterone, at least in part, due to a constituitively active form of PKA and an enhanced phosphorylation state of StAR. During luteolysis PGF2alpha of uterine origin reduces the secretion of progesterone from the corpus luteum, but intraluteal production of PGF2alpha is required for normal luteolysis. Binding of PGF2alpha to receptors on large luteal cells stimulates the secretion of oxytocin which appears to activate PKC and may also inhibit steroidogenesis in small luteal cells. PGF2alpha also activates COX-2 in large luteal cells which leads to secretion of PGF2alpha. Once intraluteal concentrations of progesterone have decreased, oxytocin binding to its receptors on small luteal cells also results in increased levels of intracellular calcium and presumably apoptosis. Increased secretion of PGF2alpha from large luteal cells activates calcium channels which likely results in apoptotic death of this cell type.

Neuroprotection by estradiol: A role of aromatase against spine synapse loss after blockade of GABA A receptors

Estrogen has been suggested to be pro-epileptic by reducing GABA synthesis, resulting in increased spine density and a decreased threshold for seizures in the hippocampus, which, once they occur, are characterized by a dramatic spine loss in the affected brain areas. As considerable amounts of estradiol are synthesized in the hippocampus, in this study we focused on aromatase, the rate-limiting enzyme in estrogen synthesis in order to examine the role of locally synthesized estrogens in epilepsy. To this end, we first examined the effects of letrozole, a potent aromatase inhibitor, on GABA metabolism in single interneurons of hippocampal dispersion cultures. Letrozole downregulated estradiol release into the medium, as well as glutamate decarboxylase (GAD) expression and GABA synthesis, and decreased the number of GAD positive cells in the cultures. Next, we counted spine synapses and measured estradiol release of hippocampal slice cultures, in which GABA A receptors had been blocked by bicuculline, in order to mimic epileptic activity. Treatment of slice cultures with bicuculline resulted in a dramatic decrease in the number of spine synapses and in a significant suppression of estrogen synthesis. The decrease in synapse number in response to bicuculline was restored by combined application of estradiol and bicuculline. Surprisingly, estradiol alone had no effect on either spine synapse number or on GAD expression and GABA synthesis. “Rescue” of synapse number in “epileptic slices” by estradiol and maintenance of GABA metabolism by hippocampus-derived estradiol points to a neuroprotective role of aromatase in epilepsy. Re-filling of estradiol stores after their depletion due to overexcitation may therefore add to therapeutical strategies in epilepsy.

Transdermal Delivery of Estradiol and Norethindrone Acetate: Effect of Vehicles and Pressure Sensitive Adhesive Matrix

Addition of 30% propylene glycol was required to maintain sink condition in the evaluation of percutaneous absorption of estradiol and norethindrone acetate. The permeability of estradiol was higher in silicone and SIS adhesives. However, estradiol was crystallized in silicone, SIS, and SBS adhesive matrix. The permeability ratio of estradiol or norethindrone acetate from acrylic pressure sensitive adhesives varied widely depending on the functional group of the acrylic adhesives. PEO grafting to acrylic adhesive seemed to change physicochemical property of acrylic adhesive and increased the permeability of estradiol and norethindrone acetate significantly. On the contrary, highly cross-linked enhancer compatible acrylic adhesive decreased the permeability of both estradiol and norethindrone acetate. 20 provided the highest enhancing effect on the permeability of both estradiol and norethindrone acetate followed by oleic acid and EP40. The permeability of the drugs from the developed system was comparable to that from commercial , although significantly lower amount of estradiol and norethindrone acetate were loaded in the developed system.

258. Characterisation of ovarian follicular growth in the brushtail possum

The number and size of follicles selected for ovulation differ between species. The aim of this study was to characterise antral follicular growth and determine the size when selection of the ovulatory follicle occurs in the monovular brushtail possum. For this study, antral follicles ≥ 1 mm were dissected from the ovaries of 31 adult female possums at different reproductive states and follicular fluid and granulosa cells were harvested from each individual follicle. Selection of the ovulatory follicle in the brushtail possum occurred when follicles reached between 2.5 and 2.8 mm in diameter. Based on the analysis of steroids in follicular fluid, before selection, most follicles produced varying amounts of oestradiol (E2), but very few if any produced progesterone (P4). After selection, the selected follicle continued to produce increasing amounts of E2 and P4, whereas most other follicles were steroidogenically inactive. Near the time of ovulation, presumably after the LH surge, P4 became the predominant steroid produced by the selected follicle and most other follicles once again produced varying amounts of E2. The number of granulosa cells per follicle was highly variable, but tended to increase with increasing diameter. Cell viability was very high, averaging about 95%. Interestingly, the morphology of granulosa cells changed markedly after selection becoming larger and granular in appearance. The weights of the vaginal cul-de-sac and uteri correlated well with the presence of a selected follicle. In ovaries from pregnant animals (n = 3), follicles grew up to 3.5 mm, and although they reached the size of a selected follicle during the follicular phase, E2 production by the other follicles was not suppressed and the weights of the cul-de-sac were less than those from non-pregnant animals with similar sized follicles. During anoestrus (n = 4), follicles did not grow beyond 2 mm and produced very little steroids.

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.

Effect of dose on the absorption of estradiol from a transdermal gel

Objectives: To study whether dose adjustments in transdermal estradiol gel treatment would result in proportional changes in estradiol bioavailability and concentrations. Methods: In an open study, 23 healthy postmenopausal women were treated consecutively with 0.5, 1.0 and 1.5 mg estradiol daily as a transdermal gel. Each dose was given for 16 days. Venous blood samples for serum estradiol and estrone measurements with RIA were taken at steady state on the 16th study day. From these concentrations, pharmacokinetic parameters for estradiol were calculated and corrected to correspond to equal dose by dividing the values by the dose. Results: Area under the estradiol time–concentration curve and peak estradiol level increased linearily and dose-proportionally with daily estradiol doses of 0.5–1.5 mg. This was shown by lack of significant differences in the dose-corrected parameters. However, the 90% confidence intervals between the doses were outside the commonly accepted levels for bioequivalence. Peak estradiol level was clearer and occurred earlier with the highest 1.5 mg estradiol dose, while more stable estradiol levels were seen with the lowest 0.5 mg estradiol dose. Conclusions: The amount of estradiol on a certain skin area seems to be the determining factor in absorption. With higher estradiol doses, the absorption will be accelerated with a clearer peak estradiol level. The linear and dose proportional absorption indicates that flexible dose adjustments within the dose range of 0.5–1.5 mg estradiol daily can be made with an anticipated effect in estradiol bioavailability and concentrations.

The role of luteal steroid hormones in the regulation of the estrous cycle of high fecundity Olkuska sheep.

The study was undertaken to investigate the steroid hormone production by sheep luteal cells. Corpora lutea were collected from 30 Olkuska sheep on Days 3, 6, 9, 12 and 15 of the estrous cycle during the reproductive season. In Experiment 1, steroid hormone concentration was estimated in extracts of CL. In Experiment 2, luteal cells were cultured in vitro for 24 h. Luteal cells isolated on Days 9 and 12 secreted high amounts of progesterone and androgens but smaller amounts of estradiol. Concentration of these steroids in CL extracts collected on the same days showed the same trend. In CL harvested on Day 15, a decrease in androgens and progesterone as well as a significant increase in estradiol were observed in culture media and in extracts. Judging from the high amounts of estradiol and low amounts of androgen observed at the end of the luteal phase, we speculate that the steroid hormones secreted by the regressing CL may play an active role in the regulation of the estrous cycle in the Olkuska sheep with autocrine influence on the luteal activity or a possible paracrine action on follicular growth. In the third Experiment, the possibility of heterogeneity in the multiple corpora lutea population of prolific Olkuska sheep was investigated. Differences were found in the level of progesterone and estradiol secretion by individual corpora lutea recovered from the same animal, which also varied in terms of weight. This is the first study which shows the existence of intra-ovarian and individual heterogeneity between corpora lutea recovered from ewes during the normal estrous cycle.