Time-release Pellets Research Articles

Long-term normalization of calcineurin activity in model mice rescues Pin1 and attenuates Alzheimer’s phenotypes without blocking peripheral T cell IL-2 response

BackgroundCurrent treatments for Alzheimer’s disease (AD) have largely failed to yield significant therapeutic benefits. Novel approaches are desperately needed to help address this immense public health issue. Data suggests that early intervention at the first stages of mild cognitive impairment may have a greater chance for success. The calcineurin (CN)-Pin1 signaling cascade can be selectively targeted with tacrolimus (FK506), a highly specific, FDA-approved CN inhibitor used safely for > 20 years in solid organ transplant recipients. AD prevalence was significantly reduced in solid organ recipients treated with FK506.MethodsTime release pellets were used to deliver constant FK506 dosage to APP/PS1 mice without deleterious manipulation or handling. Immunofluorescence, histology, molecular biology, and behavior were used to evaluate changes in AD pathology.ResultsFK506 can be safely and consistently delivered into juvenile APP/PS1 mice via time-release pellets to levels roughly seen in transplant patients, leading to the normalization of CN activity and reduction or elimination of AD pathologies including synapse loss, neuroinflammation, and cognitive impairment. Pin1 activity and function were rescued despite the continuing presence of high levels of transgenic Aβ42. Indicators of neuroinflammation including Iba1 positivity and IL-6 production were also reduced to normal levels. Peripheral blood mononuclear cells (PBMC) obtained during treatment or splenocytes isolated at euthanasia activated normally after mitogens.ConclusionsLow-dose, constant FK506 can normalize CNS CN and Pin1 activity, suppress neuroinflammation, and attenuate AD-associated pathology without blocking peripheral IL-2 responses making repurposed FK506 a viable option for early, therapeutic intervention in AD.

Unexpected long-term retention of subcutaneous beeswax implants and additional notes on dose and composition from four testosterone implant studies

Experimental manipulations of testosterone have advanced our understanding of the hormonal control of traits across vertebrates. Implants are commonly used to supplement testosterone and other hormones to organisms, as they can be readily scaled to produce desired hormone levels in circulation. Concerns about pharmacological (i.e. unnatural) doses of traditional silastic implants led to innovation in implant methods, with time-release pellets and beeswax implants proposed as solutions. A study comparing silastic, time-release pellets, and beeswax implants found the latter to be most effective in delivering a physiologically relevant dose. One proposed advantage to subcutaneous beeswax implants is that they are expected to degrade within the body, thus removing the obligation to recapture implanted individuals in the field. However, few studies have reported on dosage and no published literature has examined the assumption that beeswax implants readily degrade as expected. Here we present time-release androgen data in relation to implants containing varying levels of testosterone from four separate implant studies. In addition, we report long-term persistence of subcutaneous implants, including two cases of implants being retained for > 2 years. Finally, we offer recommendations on the composition and implementation of beeswax implants to aid the pursuit of minimally invasive and physiologically relevant manipulations of circulating hormones.

Application of Factorial Design in the Premixing Operation of Diltiazem Timed-Release Pellets

Application of Factorial Design in the Premixing Operation of Diltiazem Timed-Release Pellets

OR09-03 Brain Aromatase Is Essential for Regulation of Sexual Activity in Male Mice

Introduction: The biologically active form of estrogen, estradiol (E2), has important organizational roles in brain development and activational roles in adult brain physiology and behavior. It has been proposed that E2 formation in the brain might regulate sexual activity in various species. The mechanisms that link estrogen formation in the brain and sexual behavior, however, remain unclear. Aromatase is the key enzyme that catalyzes the conversion of testosterone (T) to E2 in the testis and brain of male mice. To determine the role of brain aromatase in male sexual activity, we generated a brain-specific aromatase knockout (bArKO) mouse model. Additionally, a newly generated total aromatase knockout (tArKO) mouse model served as a positive control. Methods: We generated the floxed aromatase mice (Aromfl/fl), which flanked the transcription and translation start sites and the common splice acceptor site for the upstream brain promoter I.f of the aromatase gene. We then crossed Nestin-Cre mice with Aromfl/fl mice to generate bArKO mice. Using the same Aromfl/fl mice, we bred tArKO via crossing with ZP3-Cre mice. Circulating and tissue (brain and testis) E2 levels were measured using liquid chromatography-tandem mass spectrometry. We assessed sexual activity in 12-14 week-old bArKO, tArKO and littermate control males over two 30-minute trials. The interactions were monitored and videotaped, and the videotape was scored for the sexual activity. To investigate whether the lack of estrogen production in the brain was causative for altered sexual behavior, 20 bArKO and 20 control mice were castrated at ~nine weeks of age and supplemented with exogenous sex hormone via 60-day time release pellet implantation. Results: E2 levels are significantly decreased in the brain but not the testis of bArKO mice as compared to control mice (P < 0.05, n=6-12). As expected, E2 levels in the brain and testis are significantly lower in tArKO mice compared with their WT littermates (n=6-9). Furthermore, we demonstrate that local aromatase expression and estrogen production in the brain is required for male sexual behavior and sex hormone homeostasis. Male bArKO mice exhibited significantly decreased sexual activity in the presence of strikingly elevated circulating T (n=5). In castrated adult bArKO mice, administration of E2 together with T restored maximum sexual behavior (n=5). Thus, aromatase in the brain is necessary for T-dependent male sexual activity. We also found that brain aromatase is required for negative feedback regulation of circulating T of testicular origin. Conclusion: Our findings suggest T activates male sexual behavior in part via conversion to E2 in the brain and provide the foundation for inhibition or enhancement of brain aromatase enzyme activity and/or utilization of selective estrogen receptor modulators in modifying sexual behavior.DCB and HZ contributed equally to this work.

A potent chemopreventive effect of diethyl ether extract of lotus peregrinus L. on apoptosis associated proteins and oxidative stress markers in mammary tumor –induced in rats

extract and to determine whether the diethyl ether extract of Lotus peregrinus L. exerts chemo-protected effect by stimulating apoptosis during medroxyprogesterone acetate (MPA)-DMBA mammary carcinogenesis using the expression of the apoptosis-associated proteins Bax and caspases 9; as well as improvement of oxidative status of the mammary tumor tissue. Sixty female wistar rats (52 days old) were divided into four equal groups. Group I (normal group): rats administered distilled water. Group II (carcinogenic group); on day 0, wistar rats were given DMBA (2.5 mg/ rat; orally) followed by second dose DMBA (2.5 mg/ rat; orally) on day 15, 90-day timed-release pellets containing 25 mg MPA were implanted’ into rats on day 30. Group III (diethyl ether protected), on day 0, wistar rats were given DMBA (2.5 mg/ rat; orally) followed by second dose DMBA (2.5 mg/ rat; orally) on day 15, on day 26 rats were administered diethyl ether (50 mg /kg. b. wt.; orally), 90-day timed-release pellets containing 25 mg MPA were implanted into rats on day 30. Group IV (diethyl ether treated group); given diethyl ether (50 mg /kg. b. wt.; orally) after induction of mammary cancer. Our findings showed the positive treatment of mammary tumor and improved oxidative status of the tissue was evident by increase in the levels of GSH, Catalase and decreased levels of serum MDA levels which in turn is an indicator of reduced oxidative stress of the tissue. Therefore, this study suggests that diethyl ether extract of Lotus peregrinus can be useful for the treatment as well as improvement of oxidative status of the mammary tumor tissue .Also, was more effective in inhibiting DMBA-MPA induced mammary tumors and modulating the expression of apoptosis associated proteins.

Sardine proteins (Sardina pilchardus) combined with green lemon zest (Citrus latifolia) improve blood pressure, lipid profile and redox status in diabetic hypertensive rats

PurposeThis paper aims to study the effect of green lemon zest combined with sardine proteins in diabetic hypertensive rats (DHRs).Design/methodology/approachMale Wistar rats (n = 30) weighing 250 ± 10 g were divided into five groups. The first group consumed a diet containing 20 per cent casein (C). The other four groups are rendered diabetic by intraperitoneal injection of streptozotocin (40 mg/kg body weight), then hypertensive by subcutaneous implantation controlled time-release pellet containing ouabain (0.25 mg/pellet). One untreated group (DHR) consumed 20 per cent casein and the three other groups consumed the same diet supplemented with 2 per cent green lemon zest (DHR-lz), or with 20 per cent of sardine protein (group DHR-sp) or with the combination of both sardine proteins and green lemon zest (group DHR-sp + lz).FindingsDHRs feeding on the combination of both sardine protein (sp) and lemon zest (lz) induced a significant decrease of diastolic blood pressure and heart rates values compared with DHR (p < 0.05). The HDLC values were increased by +55 per cent in DHR-sp + lz compared with DHR group. Moreover, plasma non-HDLC concentrations were decreased significantly compared to DHR, DHR-lz, DHR-sp and C groups. In DHR-sp + lzvs DHR group, TBARS values were decreased by −25 per cent in the liver. Moreover, kidney TBARS were significantly reduced by −66, −51, −65 and −67 per cent compared with C, DHR, DHR-lz and DHR-sp, respectively.Originality/valueThese results suggest that consumption of green lemon zest combined with sardine proteins can reduce blood pressure and tissue oxidative damage and, therefore, help to prevent cardiovascular complications in hypertensive diabetic patients.

Glucocorticoid‐Receptor Mediated Signaling Regulates Lipolysis in Northern Elephant Seal Pups

Northern elephant seals (NES) experience prolonged 2–3 month fasting periods, relying primarily on the oxidation of fatty acids to meet their energetic needs (RQ=0.71). Although fasting periods are characterized by an increase in circulating cortisol (F) and non‐esterified fatty acids (NEFA's), the functional relevance such increase in cortisol in NES is not defined. We hypothesized that F lipolysis is regulated through glucocorticoid receptor signaling (GR) in NES. The contributions of F and the GR were assessed by exogenous infusions of ACTH and/or concurrent blockade of the GR in the following groups: (1) untreated control, (2) ACTH (80 units intramuscular injection (IM)) and (3) ACTH + GR blocker (RU486) (80 units IM + 400mg RU486 in time release pellets). Plasma, and adipose and muscle biopsies were collected at days 0 (T0; immediately prior to infusion) and 6 (T6). Mean plasma NEFA levels increased 38% (0.84 ± 0.13 vs 1.16 ± 0.06mM) from T0 to T6 with ACTH infusion suggesting glucocorticoids contribute to increased lipolysis. ACTH infusion increased mean adipose HSP90 expression by 92%±18.2 while decreasing mean muscle HSP90 expression by 39%±14.5 from T0 to T6 suggesting that HSP90 may participate in GR‐mediated lipolysis in adipose while contributing to conserving lipid sources in muscle. Mean muscle CD36 expression decreased 46%±12.2 from T0 to T6 with ACTH infusion suggesting that there is a reduced intake of NEFA's by skeletal muscle, contributing to increased circulating NEFAs during lipolysis. These data suggest that the fasting‐associated increase in GR signaling is imperative for sustaining elevated levels of lipolysis during the post weaning fast, and is dependent upon the functional role of cortisol and HSP90.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 796: Reproductive hormones modulate cardioprotection from doxorubicin-induced cardiomyopathy in female spontaneously hypertensive rats

Abstract Doxorubicin (Dox) causes cardiomyopathy, partially driven by the excess generation of mitochondrial reactive oxygen species (ROS). The incidence of Dox-induced cardiac stress is greater in prepubescent and postmenopausal females compared to reproductively mature women, suggesting that reproductive hormones may mediate cardioprotection. We investigated the role of exogenous hormone replacement therapy (HRT) and naturally cycling reproductive hormones in providing cardioprotection from Dox chemotherapy. Mammary adenocarcinoma SST-2 cells were implanted in spontaneously hypertensive rats (SHRs) that were either ovariectomized and received HRT, or left reproductively normal and maintained natural hormone cycling. HRT animals received time-release pellets containing 17-β-estradiol (E2), progesterone (P4), Tamoxifen (Tam) or combinations thereof, and were subsequently treated with Dox. Naturally cycling animals were treated with Dox, the iron chelator Dexrazoxane (Drz), or Dox+Drz. Tumor size was used to evaluate anticancer activity. Vaginal cytologies and circulating E2 and P4 levels were obtained. Serum cardiac troponin I (cTnI), histopathology and echocardiograms were used to assess cardiac health. Dox treated animals supplemented with E2 and P4 (alone) showed cardioprotection relative to the Dox treated animals. Dox+/-Drz exhibited sustained anti-tumor activity independent of endocrine status. After 13 days with Dox, all naturally cycling animals arrested in diestrus (lowest hormone levels). These results demonstrate that E2 and P4 have a role in providing cardioprotection from Dox therapy in SHRs, and that estrous cycle stage may provide a safety treatment window, potentially reducing the risk of cardiomyopathy in reproductively normal females. We are also currently analyzing RNA sequencing data obtained from heart tissues to achieve a better understanding of the molecular mechanisms of cardioprotection. Citation Format: Kaytee Pokrzywinski, Elliot T. Rosen, Julia L. Bonanno, Baikuntha Aryal, Thomas Biel, Delaram Moshkelani, Steven Mog, Ashutosh Rao. Reproductive hormones modulate cardioprotection from doxorubicin-induced cardiomyopathy in female spontaneously hypertensive rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2017-796

Abstract 4639: Reproductive hormone levels modulate doxorubicin induced cardiomyopathy in female tumor-bearing spontaneously hypertensive rats

Abstract The anthracycline doxorubicin (dox) is known to cause cardiomyopathy, at least in part, through the generation of excess reactive oxygen species (ROS) in the mitochondria. Cardiomyocytes have higher concentrations of mitochondria and lower concentrations of antioxidants, making them more sensitive to excessive ROS. The incidence of dox induced cardiac stress is greater in pre-pubescent and post-menopausal females compared to age-matched males but lower in women of reproductive age. Previous studies have associated gender and age differences in cardiosensitivity with estrogen production. This is the first part of a two part study investigating the role that reproductive hormones (estrogen and progesterone) play in dox induced cardiotoxicity in female, tumor-bearing spontaneously hypertensive rats (SHRs). In this portion of the study our aim was to evaluate the effect of hormone therapy on cardioprotection from dox. Reproductively mature ovariectomized animals were implanted with time-release pellets containing a “carrier-matrix” (vehicle), 17-β-estradiol (E2), progesterone (P4), Tamoxifen (Tam) or combinations thereof. The animals were implanted with cells from an SHR-derived syngeneic breast cancer cell line (SST-2). Tumor-bearing animals were then treated with a single dose of saline or dox and monitored for 12 days. Daily vaginal cytologies were obtained to measure estrous cycle status. Serum was collected throughout the study to correlate estrous phase to E2 and P4 levels and evaluate serum protein cTnI as a biomarker for cardiac injury. In all hormone groups, dox exhibited significant anti-tumor activity and resulted in a net decrease in growth rate compared to saline controls. After 12 days exposure, animals treated with dox and implanted with Tam, Tam+E2 or vehicle pellets showed a significant increase in cTnI, indicative of cardiac damage. Dox treated animals with pellets containing E2, P4 or the combination showed a significant decrease in cTnI, demonstrating potential cardioprotection from reproductive hormone therapy. Furthermore, histopathology of cardiac sections from dox-treated groups displayed lower cardiomyopathy scores in animals that received E2, P4 or the combination of the two. We are currently evaluating additional endpoints for cardiotoxicity and cellular stress to assess the mechanism behind E2 and P4 cardioprotection from chemotherapy. Overall, our findings suggest that the hormones estrogen and progesterone reduce dox-induced cardiotoxicity. Citation Format: Kaytee L. Pokrzywinski, Elliot T. Rosen, Julia Bonanno, Thomas Biel, Delaram Moshkelani, Baikuntha Aryal, Steven Mog, V. Ashutosh Rao. Reproductive hormone levels modulate doxorubicin induced cardiomyopathy in female tumor-bearing spontaneously hypertensive rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4639.

Glucocorticoid Receptor‐mediated Lipolysis Increases following Chronic Adrenocorticotropin Infusion in Northern Elephant Seals Pups through its Imperative Interaction with Heat Shock 90 Chaperone Protein

Northern elephant seal (NES) pups experience a prolonged 2–3 month fasting period and rely primarily on the oxidation of fatty acids to meet its energetic needs. Heat shock 90 chaperone protein (HSP90) is a fundamental part of the glucocorticoid‐cortisol (GR) complex, inducing an up regulation in GR‐mediated lipolysis upon chronoic adrenocorticotropin infusion. This fast is characterized by an increase in cortisol, HSP 90 and non‐esterified fatty acids (NEFA's). However, the functional relevance of this increase in cortisol via the GR is not well defined. We hypothesized that ACTH infusion increases lipolysis in NES. The contributions of cortisol and its receptor (GR) were assessed by exogenous infusion of ACTH and/or concurrently blocking the glucocorticoid receptor in the following groups: 1) control (no treatment), 2) ACTH (40 units of ACTH gel), 3) GR‐blocker (400mg mifepristone in time release pellets), and 4) ACTH+GR‐blocker. Plasma and adipose biopsy samples were collected at days 0 (T0; immediately prior to infusion) and 6 (T6). Mean plasma cortisol concentrations decreased 41±5% in the ACTH‐infused group and 21±3% in the combo group, but remained constant in the control and GR‐blocker groups suggestive of adrenal exhaustion. In contrast, mean plasma aldosterone increased 135%± 4 in the ACTH‐infused group suggesting that the adrenal gland in NES pups is differentially responsive (zona fasciculata vs zona glomerulosa). Mean NEFA levels increased 38%±4 in the ACTH‐infused group suggesting that ACTH‐mediated effects contribute to increased lipolysis. Mean heat shock protein 90 levels increased 119±1 in the ACTH infused group, 31±3 in the GR‐blocker and 192± 4 in the ACTH+GR‐Blocker group suggesting that mifepristone has no residual effect on the binding of HSP90 to the glucocorticoid receptor, making this interaction imperative in GR‐mediated lipolysis. Collectively, this data suggests that the fasting‐associated increase in cortisol is an important contributing factor in stimulating lipolysis potentially through the glucocorticoid receptor.

Short-term, high-dose administration of corticosterone by injection facilitates trace eyeblink conditioning in young male rats

Glucocorticoids released as part of the physiological response to stress are known to affect cognitive function, presumably via effects on the hippocampus. Trace classical eyeblink conditioning is an associative learning task which depends on the hippocampus and has been used to examine the development of learning processes in young mammals. Previously, we demonstrated deficits in trace eyeblink conditioning associated with postnatal administration of the glucocorticoid corticosterone by creating a sustained elevation with methods such as subcutaneous timed-release pellets and osmotic mini-pumps which were active over several days. In the present study, we examined the effects of an oscillating pattern of corticosterone elevation on subsequent trace eyeblink conditioning. Twice daily corticosterone injections (high, low, or vehicle) were administered over a 3-day period, starting at postnatal day 15. Then, on postnatal day 28, animals underwent trace classical eyeblink conditioning to examine the possible influence of earlier corticosterone elevations on the development of learning and memory. Eyeblink conditioning was affected by corticosterone treatments, but only for males, and only very early in acquisition; Males receiving the high dose of corticosterone exhibited facilitation of learning relative to controls. These data demonstrate that oscillating corticosterone elevations produce opposite effects on this associative learning task than do sustained elevations.

Towards more physiological manipulations of hormones in field studies: Comparing the release dynamics of three kinds of testosterone implants, silastic tubing, time-release pellets and beeswax

Hormone manipulations are of increasing interest in the areas of physiological ecology and evolution, because hormones are mediators of complex phenotypic changes. Often, however, hormone manipulations in field settings follow the approaches that have been used in classical endocrinology, potentially using supra-physiological doses. To answer ecological and evolutionary questions, it may be important to manipulate hormones within their physiological range. We compare the release dynamics of three kinds of implants, silastic tubing, time-release pellets, and beeswax pellets, each containing 3mg of testosterone. These implants were placed into female Japanese quail, and plasma levels of testosterone measured over a period of 30days. Testosterone in silastic tubing led to supraphysiological levels. Also, testosterone concentrations were highly variable between individuals. Time-release pellets led to levels of testosterone that were slightly supraphysiological during the first days. Over the period of 30days, however, testosterone concentrations were more consistent. Beeswax implants led to a physiological increase in testosterone and a relatively constant release. The study demonstrated that hormone implants in 10mm silastic tubing led to a supraphysiological peak in female quail. Thus, the use of similar-sized or even larger silastic implants in males or in other smaller vertebrates needs careful assessment. Time-release pellets and beeswax implants provide a more controlled release and degrade within the body. Thus, it is not necessary to recapture the animal to remove the implant. We propose beeswax implants as an appropriate procedure to manipulate testosterone levels within the physiological range. Hence, such implants may be an effective alternative for field studies.

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus)

Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein ‘progesterone receptor membrane component 1’ (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53pmol/g testis and 0.22pmol/g testis, and Bmax 1.8 and 5.7nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.

Antiandrogen flutamide protects male mice from androgen-dependent toxicity in three models of spinal bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a late-onset, progressive neurodegenerative disease linked to a polyglutamine (polyQ) expansion in the androgen receptor (AR). Men affected by SBMA show marked muscle weakness and atrophy, typically emerging midlife. Given the androgen-dependent nature of this disease, one might expect AR antagonists to have therapeutic value for treating SBMA. However, current work from animal models suggests otherwise, raising questions about whether polyQ-expanded AR exerts androgen-dependent toxicity through mechanisms distinct from normal AR function. In this study, we asked whether the nonsteroidal AR antagonist flutamide, delivered via a time-release pellet, could reverse or prevent androgen-dependent AR toxicity in three different mouse models of SBMA: the AR97Q transgenic (Tg) model, a knock-in (KI) model, and a myogenic Tg model. We find that flutamide protects mice from androgen-dependent AR toxicity in all three SBMA models, preventing or reversing motor dysfunction in the Tg models and significantly extending the life span in KI males. Given that flutamide effectively protects against androgen-dependent disease in three different mouse models of SBMA, our data are proof of principle that AR antagonists have therapeutic potential for treating SBMA in humans and support the notion that toxicity caused by polyQ-expanded AR uses at least some of the same mechanisms as normal AR before diverging to produce disease and muscle atrophy.

SINGLE-TREATMENT PORCINE ZONA PELLUCIDA IMMUNOCONTRACEPTION ASSOCIATED WITH REDUCTION OF A POPULATION OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS)

Previous reports have demonstrated gradual reductions of white-tailed deer (Odocoileus virginianus) populations through immunocontraception, with stabilization occurring after 2-4 yr of treatment, and subsequent reductions of 6-10% annually. These studies employed porcine zona pellucida (PZP) vaccines that required two initial treatments and annual retreatments. From 2005 to 2010, 258 adult and yearling female deer on Fripp Island, South Carolina, were treated with one of several PZP preparations designed to produce 2+ yr of effective contraception with a single treatment. These included several preparations of SpayVac and of native PZP-adjuvant emulsion plus PZP and QA-21 in timed-release pellets. Deer were chemically immobilized, ear-tagged, and administered initial treatments by hand in February-March. Some treated deer were boosted remotely with PZP-adjuvant emulsion 1.5 - 4.5 yr after initial treatments. Ground-based distance sampling was used to estimate deer population density at Fripp Island, a resort community, and at a relatively undeveloped neighboring control site, Hunting Island. Most vaccine preparations tested reduced fawning rates by 75% to 95% for at least 1 yr. From 2005 to 2011, deer density on Fripp Island declined by 50%, from 72 deer/km(2) to 36 deer/km(2), an average annual reduction of 11%. In contrast, population density on the Hunting Island control site fluctuated between 2005 and 2011, averaging 23 deer/km(2) (range, 19-28 deer/km(2)). Population declines on Fripp Island were associated with an increase in the proportion of treated females and with a progressive decrease in winter fawn:doe ratios, from 1.21 fawns/doe in 2005 to 0.19 fawns/doe in 2010. Winter fawn:doe ratios averaged 1.36 fawns/doe (range, 0.84 - 1.62 fawns/doe) at the Hunting Island control site. Annual survivorship averaged approximately 79% among ear-tagged females. The rate at which deer populations diminished in association with PZP treatments on Fripp Island was higher than that seen at other study sites, although the reasons for the more rapid decline on Fripp Island are not well understood.

319 REDUCED SUPEROVULATION EFFICIENCY BY HIGH-DOSE TREATMENT OF DEHYDROEPIANDROSTERONE IN MICE

Strain differences in in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse oocytes, we examined the effect of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, in female mice on oocyte fertilizability. The DHEA pellets (0.25, 1.5, or 5 mg pellet–1, 21-day release form, Innovative Research of America, Sarasota, FL, USA) or corresponding placebo pellets were implanted subcutaneously in 9-week-old female 129X1/SvJJmsSlc mice. On Day 18 of implantation, superovulation was induced in these females by injections of pregnant mare serum gonadotropin (PMSG) and hCG 48 h apart. On Day 21, IVF was conducted using oocytes collected from the oviducal ampullae of these females and epididymal sperm from Slc : ICR male mice using TYH medium. Then, the embryos were cultured in vitro in Whitten’s medium for 96 h. Plasma steroid levels and expression of 5 ovarian proteins (PTEN and receptors for FSH, androgen, oestrogen, and progesterone) at oocyte collection were measured by enzyme immunoassay and quantitative Western blots, respectively. Embryo development into 2-cell and blastocyst stages at each dose at 24 h and 96 h after insemination, respectively, were compared between DHEA and placebo groups using weighted ANOVA with angular transformations. Other observed values were compared using Student’s t-test. Treatment with DHEA suppressed the numbers of ovulated oocytes in the 1.5 and 5 mg groups (DHEA v. placebo: 21.0 ± 2.6 v. 32.5 ± 2.8 and 19.9 ± 1.4 v. 27.1 ± 1.6, respectively, n = 10; P &lt; 0.05), but not in the 0.25 mg group (26.6 ± 3.2 v. 24.8 ± 2.4). Treatment with DHEA did not affect the percentages of 2-cell embryo formation at any dose, ranging 50 to 60%. In the 0.25 mg group, DHEA treatment tended to increase blastocyst formation rate (1.8 ± 0.8% v. 0.4 ± 0.4%; not significant). However, the treatment at 1.5 mg suppressed the rate (0.0 ± 0.0% v. 3.1 ± 0.7%; P &lt; 0.05) and treatment at 5 mg did not affect the rate (1.3 ± 0.9% v. 0.8 ± 0.6%). Plasma testosterone levels were increased by DHEA at 1.5 and 5 mg (338.2 ± 39.8 v. 197.0 ± 8.9 pg mL–1 and 534.9 ± 111.4 v. 241.8 ± 34.4 pg mL–1, respectively, n = 6; P &lt; 0.05), but not at 0.25 mg (247.4 ± 22.0 v. 252.5 ± 35.6 pg mL–1, n = 6). No significant difference was induced by DHEA in plasma DHEA, progesterone, or oestradiol at any doses. Ovarian PTEN protein was more abundant in DHEA group than in placebo group at 5 mg (P &lt; 0.05), tended to be more abundant at 1.5 mg (P ≈ 0.14), and was not different at 0.25 mg (P ≈ 0.35). The amounts of the 4 receptor proteins were not significantly changed by DHEA at any dose. These results suggest that DHEA at a low dose (e.g. 0.25 mg pellets for 21 days) might have a potential to improve in vitro fertilizability of mouse oocytes. Higher doses of DHEA reduced superovulation efficiency, perhaps because of the high testosterone level induced by the high-dose treatment of DHEA. The high testosterone level might upregulate ovarian PTEN expression, which might suppress ovarian primordial follicle activation. A more detailed study is needed to determine the optimal dose, timing, and duration of DHEA treatment for the improvement of female fertilizability.

Dose-dependent effects of calorie restriction on gene expression, metabolism, and tumor progression are partially mediated by insulin-like growth factor-1.

The prevalence of obesity, an established risk and progression factor for breast and many other cancer types, remains very high in the United States and throughout the world. Calorie restriction (CR), a reduced-calorie dietary regimen typically involving a 20–40% reduction in calorie consumption, prevents or reverses obesity, and inhibits mammary and other types of cancer in multiple tumor model systems. Unfortunately, the mechanisms underlying the tumor inhibitory effects of CR are poorly understood, and a better understanding of these mechanisms may lead to new intervention targets and strategies for preventing or controlling cancer. We have previously shown that the anticancer effects of CR are associated with decreased systemic levels of insulin-like growth factor-1 (IGF-1), the primary source of which is liver. We have also reported that CR strongly suppresses tumor development and growth in multiple mammary cancer models. To identify CR-responsive genes and pathways, and to further characterize the role of IGF-1 as a mediator of the anticancer effects of CR, we assessed hepatic and mammary gland gene expression, hormone levels and growth of orthotopically transplanted mammary tumors in control and CR mice with and without exogenous IGF-1. C57BL/6 mice were fed either control AIN-76A diet ad libitum (AL), subjected to 20%, 30%, or 40% CR plus placebo timed-release pellets, or subjected to 30% or 40% CR plus timed-release pellets delivering murine IGF-1 (mIGF-1, 20 μg/day). Compared with AL-fed controls, body weights were decreased 14.3% in the 20% CR group, 18.5% in the 30% CR group, and 38% in the 40% CR group; IGF-1 infusion had no effect on body weight. Hepatic transcriptome analyses indicated that compared with 20% CR, 30% CR significantly modulated more than twice the number of genes and 40% CR more than seven times the number of genes. Many of the genes specific to the 40% CR regimen were hepatic stress-related and/or DNA damage-related genes. Exogenous IGF-1 rescued the hepatic expression of several metabolic genes and pathways affected by CR. Exogenous IGF-1 also rescued the expression of several metabolism- and cancer-related genes affected by CR in the mammary gland. Furthermore, exogenous IGF-1 partially reversed the mammary tumor inhibitory effects of 30% CR. We conclude that several genes and pathways, particularly those associated with macronutrient and steroid hormone metabolism, are associated with the anticancer effects of CR, and that reduced IGF-1 levels can account, at least in part, for many of the effects of CR on gene expression and mammary tumor burden.

Effects of selenium supplementation and chronic inflammation on bone microarchitecture and strength in mice

Timed‐pregnant C57BL6 mice were fed a Torula yeast selenium‐depletion diet from the final days of gestation through lactation. At 23 days of age, pups were weaned and randomly assigned to the depletion diet or to diets supplemented with 0.2, 2 or 4 mg/kg diet of Se added as sodium selenate for 14 weeks. At 96–98 days of age mice were randomly assigned to placebo or to lipopolysaccharide (E. coli Serotype 0127:B8) treatment to produce chronic inflammation. Time release pellets (0 or 0.1 μg/g body weight/d) were implanted subcutaneously. Body weight was reduced by LPS 14 days after implantation but differences were no longer significant by 28 days. Bone microarchitecture was evaluated in tibia and 4th lumbar vertebra (L4) and force to compress was estimated on trabecular cores using finite element analysis (Scanco μCT40). Bone volume/total volume, connectivity density, and trabecular number were significantly reduced by LPS treatment while trabecular separation was increased by LPS treatment in both bones. In spine significant interactions affected size‐independent stiffness and von Mises stresses. Overall, chronic inflammation at a level that does not produce severe body weight loss still significantly impaired measures of bone microarchitecture and strength. (Supported by Oklahoma Center for Advancement of Science and Technology).

179 EFFECTS OF DEHYDROEPIANDROSTERONE ON SPERM FERTILIZABILITY IN VITRO AND TESTICULAR GENE EXPRESSION

Strain differences of in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse sperm, we examined the effects of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, on sperm fertilizability and testicular gene expression. DHEA pellets (5 mg pellet–1, 21-day release form; Innovative Research of America) or placebo pellets were implanted subcutaneously in 9-week-old male mice from 2 strains: C57BL/6CrNSlc (B6) and 129X1/SvJJmsSlc (129X1). After 21 days, in vitro fertilization was conducted using epididymal sperm from these males and oocytes from superovulated 4-week-old Slc:ICR females. The percentages of 2-cell embryo formation in the placebo and DHEA groups were 78.2 ± 14.2% vs 89.3 ± 2.5%, respectively (mean ± standard error of the mean, n = 4 per group) using B6 sperm and 40.9 ± 8.8% vs 41.3 ± 3.1%, respectively (n = 4 per group) using 129X1 sperm, indicating no significant effect of DHEA treatment (P &gt; 0.05 by 2-way ANOVA). However, a strain difference was quite evident (P &lt; 0.05 by 2-way ANOVA). Quantitative Western blotting using testicular protein extracts with glyceraldehyde-3-phosphate dehydrogenase as an internal control showed that the expression of androgen receptor protein (AR) was significantly higher in B6 than in 129X1 males (P &lt; 0.05 by 2-way ANOVA), whereas there was no significant effect of DHEA on the amount of AR in either strain (P &gt; 0.05 by 2-way ANOVA). In B6 testes, 2-dimensional electrophoresis indicated that 1 protein spot was denser in DHEA-treated males than in placebo-treated males. In 129X1 testes, DHEA did not change the density of the corresponding spot. Mass spectrometry of the spot suggested that it was Cu/Zn superoxide dismutase (SOD1). Thus, 21-day-release pellets of 5 mg DHEA had no significant effect on sperm fertilizability in vitro in 2 strains, but the DHEA pellets induced a strain-dependent testicular protein expression, which might be because of the differential AR expression. Although DHEA treatment of male mice has the potential to improve sperm fertilizability in vitro, a more detailed study is needed to determine the optimal dose, age and duration of DHEA treatment. This work was supported by a grant from the Ministry of Health, Labour and Welfare, Japan.

Environmental exposure, estrogen and two X chromosomes are required for disease development in an epigenetic model of lupus

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6xSJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA-methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X-chromosome effects and through suppression of ERK signaling by environmental agents.