Sandra L. Gray

Vitellogenin induction in painted turtle, Chrysemys picta, as a biomarker of exposure to environmental levels of estradiol

Ponds within cattle farms often support turtle and fish populations and are impacted by manure runoff. Cattle excrete metabolized (glucuronide-conjugated) hormones in feces and urine into these ponds, and bacteria cleave the glucuronide metabolites to active steroids, which can be stable for several weeks in wastewater. The objectives of this study were to (1) assess levels of xenoestrogens found in ponds near livestock pastures; and (2) assess whether these levels of xenoestrogens induce vitellogenin (VTG) in painted turtles in the laboratory and field. We collected water twice, 6 weeks apart, and placed turtle traps weekly into two ponds, which receive runoff from beef cattle pastures, and into one pond with no cattle farm effluents. Water E(2) levels were analyzed using C(18) solid phase extraction disks and detected in a radioimmunoassay (RIA). Plasma was collected from painted turtles (Chrysemys picta) captured from these ponds and VTG levels were measured via enzyme linked immunosorbent assay (ELISA). Nine additional turtles were collected from a pond at the South Carolina Botanical Gardens, which receives no farm runoff, and were exposed in the laboratory to nominal concentrations of 0.15, 1.5, and 15 ng/l estradiol (static renewal) over a 28-day period, followed by 14 days in clean water. Plasma samples were taken weekly for VTG measurement via ELISA. Levels of free estradiol in the water column of farm ponds range from 0.05 to 1.80 ng/l, as measured by RIA, and up to 7.4 ng/l as measured by ER-beta binding affinity. This is similar to what has been reported in streams receiving sewage treatment works (STW) effluents. In the laboratory, plasma VTG in male painted turtles could not be induced even at the high E(2) dose (9.45 ng/l) after 28 days. In the field, VTG levels were induced only in females when compared with animals from the SC Botanical Gardens. Adult male turtles need to be primed with high doses of E(2) prior to being able to respond to exogenous E(2). Given that males would not typically be sensitized in the wild, environmentally relevant levels of E(2) may not be sufficient to affect them. However, higher VTG levels in females could potentially change their reproductive fitness by altering egg size or by shifting energy allocations away from other survival needs. Long-term studies are needed to study potential impacts of VTG induction on female turtle reproductive success.

The insulin-like growth factor (IGF) system and gonadotropin regulation: actions and interactions

Insulin-like growth factors (IGF) are polypeptides that regulate growth, differentiation and survival in a multitude of cells and tissues. The IGF system consists of ligands, receptors, binding proteins and binding protein proteases. The influence of the IGF system on reproductive parameters, specifically gonadotropin release and interactions between the IGF system and other effectors of gonadotropin release will be examined in this review.

Mycotoxins in Root Extracts of American and Asian Ginseng Bind Estrogen Receptors α and β

The estrogenic activity of ginseng has been the subject of conflicting reports. Cell proliferation, induction of estrogen-responsive genes, and isolated cases of adverse reactions such as postmenopausal vaginal bleeding and gynecomastia have been reported after ginseng treatment. Other studies report antiproliferative effects with no induction of estrogen-responsive genes. We developed estrogen receptor (ER) α and ERβ competitive binding assays using recombinant receptors and [3H]-17β-estradiol to detect phytoestrogens in extracts of Asian ginseng root (Panax ginseng C. A. Meyer) and American ginseng root (Panax quinquefolius L.). Root extracts contained substances that bound both receptor isoforms. These substances had a two to three times greater affinity for ERβ. Significantly higher binding was found in methanol extracts than in hot water extracts. Subsequent analysis of the extracts revealed significant ER binding attributable to zearalenone, the estrogenic mycotoxin produced by several Fusarium species. The ER showed no binding affinity for Rb1 and Rg1, the major ginsenosides found in P. quinquefolius and P. ginseng, respectively. Thus, ginseng extraction methods, plant species tested, and mycotoxin contaminants may help to explain the disparate literature reports. The prevalence and health significance of fungal contamination in herbal products used for medicinal purposes should be further investigated.

Residues from anabolic preparations after good veterinary practice.

The purpose of this study was to determine the endogenous concentrations of estrogens, particularly estradiol‐17β (E2β), in edible tissues of beef cattle (females and intact and neutered males) and the concentrations of E2β, and trenbolone beta and alpha (βTb, αTb) after an E2β and/or trenbolone acetate (TA) ear implant. Radioimmunoassays were validated for quantitation of E2β (active isomer), E2α, estrone (E1), βTb and αTb for bovine muscle, liver, kidney and fat tissues. The criteria of accuracy, precision, specificity and sensitivity were applied according to the standards of the U.S. Food & Drug Administration. In steer tissues, endogenous E2β was <15 ppt, as was heifer muscle; but heifer liver and kidney were 3‐fold greater. An E2β implant in steers had no effect on muscle E2β concentration, but increased E2β in liver and fat 4‐ and 3‐fold, respectively, but by 24 h post‐implant removal, E2β had fallen by half. Tissue E1 concentrations in cyclic females were similar to E2β, but rose many fold greater than did E2β during gestation; E2β rose 3‐fold during gestation. After E2β/TA implant, steer tissues had E2β concentrations equal to (for muscle and fat) and one‐half (for liver) the E2β measured in E2β implant only steers; βTb was in a low range (250–380 ppt) in muscle, liver and fat and αTb was even lower, except in liver (800–1500 ppt). An implant of TA only (no E2β) resulted in βTb and αTb concentrations 2–3‐fold greater in liver, kidney and fat, but no greater in muscle than βTb in tissues of E2β/TA implant steers. In conclusion, anabolic implants in steers resulted in tissue E2β concentrations less than the FDA allowable increment and βTb in the lowest quartile (0.25) of a part per billion 30 days after implant.

Effect of holding time and temperature of bovine whole blood on concentration of progesterone, estradiol-17β and estrone in plasma and serum samples☆

Bovine jugular venous blood was collected, with and without heparin, and aliquoted into 140 12-ml tubes. Four subsamples (two heparinized and two coagulated) were centrifuged immediately (time zero) and plasma or serum was aspirated and stored at -20 degrees C. One-half of the remaining subsamples were stored at 4 degrees C and the other one-half at 25 degrees C (room temperature). At 1-h intervals (0 to 24 h), 6-h intervals (24 to 72 h) and at 96 and 120 h, four subsamples (heparinized and coagulated at both 4 degrees C and 25 degrees C) were centrifuged, plasma or serum was aspirated and stored at -20 degrees C. Whole blood incubation for 1 h at 25 degrees C reduced mean plasma and serum progesterone (P(4)) concentration (P<0.05). Similarly, whole blood incubation at 4 degrees C for 2 and 3 h, respectively, reduced mean plasma and serum P(4) concentration (P<0.05). No difference was found in mean P(4) concentration between plasma and serum samples harvested from whole blood incubated at 4 degrees C or 25 degrees C. Concentration of estradiol-17beta (E(2)) and estrone (E(1)) fluctuated over time, irrespective of holding temperature. There was a blood type, heparinized or coagulated, by time interaction (P<0.01) for both E(2) and E(1) concentrations It was concluded that incubation time and temperature between collection and centrifugation of bovine blood samples influenced the assayable P(4) concentration in both plasma and serum. In contrast, incubation temperature had no effect on assayable E(2) and E(1) concentrations, but assayable E(2) and E(1) over time were differentially affected, depending on whether plasma or serum was assayed.

Physiological basis for use of insulin-like growth factors in reproductive applications: a review.

The insulin-like growth factors (IGF-I and -II) are ubiquitously expressed factors that regulate cell growth, differentiation and maintenance of differentiated cell function. All aspects of male and female reproduction are influenced by the IGF system. This review will examine the IGF system as it pertains to reproductive physiology and applications.

Effects of Panax ginseng, zearalenol, and estradiol on sperm function

Background Estrogen signaling pathways are modulated by exogenous factors. Panax ginseng exerts multiple activities in biological systems and is classified as an adaptogen. Zearalenol is a potent mycoestrogen that may be present in herbs and crops arising from contamination or endophytic association. The goal of this study was to investigate the impact of P. ginseng, zearalenol and estradiol in tests on spermatozoal function. Methods The affinity of these compounds for estrogen receptor (ER)—alpha and beta (ERα and ERβ)—was assessed in receptor binding assays. Functional tests on boar spermatozoa motility, movement and kinematic parameters were conducted using a computer-assisted sperm analyzer. Tests for capacitation, acrosome reaction (AR), and chromatin decondensation in spermatozoa were performed using microscopic analysis. Results Zearalenol—but not estradiol (E2)- or ginseng-treated spermatozoa—decreased the percentage of overall, progressive, and rapid motile cells. Zearalenol also decreased spontaneous AR and increased chromatin decondensation. Ginseng decreased chromatin decondensation in response to calcium ionophore and decreased AR in response to progesterone (P4) and ionophore. Conclusion Zearalenol has adverse effects on sperm motility and function by targeting multiple signaling cascades, including P4, E2, and calcium pathways. Ginseng protects against chromatin damage and thus may be beneficial to reproductive fitness.

Impact of kudzu and puerarin on sperm function.

The goal of this study was to investigate the impact of kudzu (Pueraria mirifica) and the isoflavone puerarin in functional toxicological tests on spermatozoa and to assess the affinity of extracts and pure isoflavones for estrogen receptor (ER)-alpha and -beta (ERα, ERβ) in receptor binding assays. Capacitation, acrosome reaction and chromatin decondensation in spermatozoa were analyzed using microscopic analysis. Kudzu, but not puerarin, reduced motility of sperm. Puerarin reduced the percent spontaneous acrosome reaction in spermatozoa. The pathways used by kudzu that affect sperm function are not fully mirrored by puerarin. Puerarin, kudzu and its other phytoestrogenic components displayed preferential affinity for ERβ, however the diverse effects of kudzu and puerarin on sperm function implicate the involvement of multiple signaling systems.

Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa

Abstract Multiple inter-connected signaling pathways, involving kinases and phosphatases, form a framework that controls sperm motility, function, and fertilizing ability. Methods that give a broad view of the proteomic landscape may prove valuable in uncovering new crosstalk connections, as well as in discovering new proteins within this regulatory framework. A multi-immunoblotting strategy was utilized to evaluate this concept on human and porcine spermatozoa samples. In human and porcine spermatozoa, a diversity of kinases were identified including protein kinase A (PKA), protein kinase B (PKB), isoforms of protein kinase C (PKC), calmodulin-dependent kinases (CAMK), casein kinase (CK), and isoforms of glycogen synthase kinase (GSK3). Several phosphatases, such as protein phosphatase (PP)-1, PP2A, PP2C, and mitogen activated protein kinase (MAPK) phosphatase (MKP-1), were identified in human spermatozoa. The phosphorylation epitopes recognized belonged to members of the MAPK family, in addition to α and β isoforms of GSK3 and cAMP response element binding protein (CREB). Proteomic approaches that allow a broad view may aid in understanding the crosstalk between signaling systems in spermatozoal physiology.

Multiple Integrated Complementary Healing Approaches: Energetics & Light for bone

A synergistic-healing strategy that combines molecular targeting within a system-wide perspective is presented as the Multiple Integrated Complementary Healing Approaches: Energetics And Light (MICHAEL). The basis of the MICHAEL approach is the realization that environmental, nutritional and electromagnetic factors form a regulatory framework involved in bone and nerve healing. The interactions of light, energy, and nutrition with neural, hormonal and cellular pathways will be presented. Energetic therapies including electrical, low-intensity pulsed ultrasound and light based treatments affect growth, differentiation and proliferation of bone and nerve and can be utilized for their healing benefits. However, the benefits of these therapies can be impaired by the absence of nutritional, hormonal and organismal factors. For example, lack of sleep, disrupted circadian rhythms and vitamin-D deficiency can impair healing. Molecular targets, such as the Wnt pathway, protein kinase B and glucocorticoid signaling systems can be modulated by nutritional components, including quercetin, curcumin and Mg(2+) to enhance the healing process. The importance of water and water-regulation will be presented as an integral component. The effects of exercise and acupuncture on bone healing will also be discussed within the context of the MICHAEL approach.