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El 17β-estradiol, una hormona clásicamente asociada a funciones reproductivas, ha adquirido trascendencia por sus efectos en varios tejidos no asociados a la reproducción. Asimismo, se ha propuesto que el 17β-estradiol contribuiría a la supervivencia celular en varios tejidos. En músculo esquelético ha sido descripta la presencia de receptores de estrógenos (ER) aunque el mecanismo molecular activado por la hormona y el rol fisiológico de la misma en este tejido no han sido totalmente dilucidados. En este laboratorio, utilizando la línea celular de músculo esquelético murino C2C12 se demostró que, sumado a la localización citosólica-nuclear, los ER se encuentran en mitocondria y fracción microsomal. En esta tesis, se investigó el rol del 17β-estradiol sobre las cascadas de señalización que se activan durante la apoptosis en mioblastos esqueléticos, caracterizando los mecanismos moleculares involucrados y enfatizando sobre la modulación de funciones mitocondriales. Los estudios realizados sugieren que la inducción de apoptosis con H2O2 en células C2C12 activa diferentes vías de señalización que incluyen la fosforilación temprana de PKD, y posteriormente las de JNK, PKCδ, PKCθ, p53 y p66Shc, efectos que son revertidos con el pretratamiento con 17β-estradiol. En particular, PKCδ actúa upstream JNK que, a su vez, conduce a la fosforilación y translocación mitocondrial de p66Shc en respuesta al H2O2. Este efecto también es contrarrestado por 17β-estradiol, resultando en la protección del potencial de membrana mitocondrial y la prevención de la apertura prolongada del poro de permeabilidad transitoria mitocondrial. Esta acción protectora del 17β-estradiol sobre la fisiología mitocondrial también incluye la inhibición de la translocación de Bax hacia mitocondria. Asimismo, se observó que uno de los sucesos en el inicio de la apoptosis disparados por H2O2 es el aumento en la formación de estructuras tipo-TNT (tunneling nanotubes) entre las células para promover la transferencia de mitocondrias sanas desde las células no estresadas a las estresadas, mientras que el pretratamiento con la hormona disminuye su formación, efecto probablemente relacionado con la reducción en la proporción de células estresadas que necesitarían revertir la disfunción mitocondrial. Estas mitocondrias disfuncionales producen mayor cantidad de especies reactivas del oxígeno y cuando sus niveles aumentan, las enzimas antioxidantes son fundamentales para contrarrestar sus efectos negativos. En esta tesis se observó que la disminución de la capacidad antioxidante inducida por H2O2 es revertida por el pretratamiento con 17β-estradiol mediante la regulación de las actividades enzimáticas y/o expresión proteica de MnSOD, GPx y CAT a través de ERs. Por otra parte, se evidenció la regulación de FoxOs por 17β-estradiol que induciría la inhibición de la transcripción de genes proapoptóticos, y además que el inductor de apoptosis regula negativamente a nivel transcripcional a Bcl-2, y positivamente a p66Shc, PUMA y PERP, acciones que son revertidas por 17β- estradiol. Por otro lado, la regulación transcripcional de MDM2 por el H2O2, que involucra a ERK, se relacionaría con la actividad de p53 y FoxO3a inactivo. Esta tesis aporta conocimientos básicos sobre los mecanismos moleculares activados por 17β-estradiol frente a la apoptosis inducida por H2O2 en células musculares esqueléticas que resultan en supervivencia celular. / 17β-estradiol, a hormone typically associated with reproductive functions, has gained significance for its effects on several tissues not associated with reproduction. Furthermore, it has been proposed that 17β-estradiol contributes to cell survival in various tissues. In skeletal muscle it has been described the presence of estrogen receptors (ER) but the molecular mechanism activated by the hormone and its physiological role over this tissue have not been fully elucidated. In this laboratory, using the murine skeletal muscle cell line C2C12 it was shown that, in addition to the cytosolic-nuclear localization, the ER was found in mitochondria and microsomal fraction. In this thesis, it was investigated the role of 17β-estradiol on the signaling cascades that are activated during apoptosis in skeletal myoblasts, characterizing the molecular mechanisms involved and emphasizing on modulation of mitochondrial functions. The studies suggest that induction of apoptosis with H2O2 in C2C12 cells activates different signaling pathways including early phosphorylation of PKD, and then the phosphorylation of JNK, PKCδ, PKCθ, p53 and p66Shc, effects that are reversed by pretreatment with 17β-estradiol. In particular, PKCδ acts upstream JNK which, in turn, leads to phosphorylation and mitochondrial translocation of p66Shc in response to H2O2. This effect is counteracted by 17β-estradiol, resulting in the protection of mitochondrial membrane potential and prevention of sustained opening of the mitochondrial permeability transition pore. This protective effect of 17β-estradiol on mitochondrial physiology also includes the inhibition of Bax translocation to mitochondria. It was also observed that one of the events in the beginning of apoptosis triggered by H2O2 is an augmented formation of TNT-like structures (tunneling nanotubes) between cells that promotes the transfer of healthy mitochondria from unstressed to stressed cells, whereas pretreatment with the hormone decreases its formation, effects that would probably be related to the reduction in the proportion of stressed cells that needs to reverse mitochondrial dysfunction. Since dysfunctional mitochondria produce more reactive oxygen species, when their levels are increased, antioxidant enzymes are essential to counteract their negative effects. In this thesis, it was observed that the decrease in H2O2-induced antioxidant capacity is reversed by pretreatment with 17β-estradiol by regulating enzyme activities and/or protein expression of MnSOD, GPx and CAT via ERs. Additionally, it was observed that the regulation of FoxOs mediated by 17β- estradiol would be related to the inhibition of transcription of proapoptotic genes, and moreover that the apoptotic inductor negatively regulates the transcriptional level of Bcl-2, and positively the transcriptional levels of p66Shc, PUMA and PERP. These actions are reversed by pretreatment with 17β-estradiol. Also, it was suggested that the transcriptional regulation of MDM2 by H2O2 involved ERK and that it could be related to p53 activity and inactive FoxO3a. This thesis provides basic information on the 17β-estradiol-activated mechanisms against H2O2-induced apoptosis in skeletal muscle cells resulting in cell survival molecular mechanisms.

Current data indicates that feeding dairy calves more nutrients in early life allows them to produce more milk in the future. Mechanisms responsible are poorly understood. Thirty-six Holstein heifer calves were fed either a restricted (R; 20.2% crude protein [CP], 19.8% fat, dry matter (DM) basis, fed at 0.44 kg/hd/d, DM basis) or an enhanced (EH; 28.9% CP, 26.2% fat, DM basis, fed at 1.08 kg/hd/day, DM basis) milk replacer (MR) and given either a placebo or estradiol (E2) implant to assess differential responses to E2. Our underlying hypothesis was that calves fed more nutrients are better able to respond to mammogenic stimuli and will have a more developed mammary gland as a result of imposed treatments. Enhanced-fed calves grew at a faster rate, were heavier at weaning, and had more functional mammary tissue (i. e., parenchyma; PAR) mass in the mammary gland at weaning (7.3-fold). Additionally, biochemical composition of the PAR was not impacted by the dietary treatments imposed. Furthermore, EH-fed calves had an increase in the number of actively dividing cells throughout the mammary PAR as well as increased intensity of estrogen receptor expression in the population of cells expressing the estrogen receptor. Enhanced-fed calves had an up-regulation of genes and pathways in the PAR related to metabolism, cellular signaling, and cellular growth. When given E2, EH-fed calves experienced the greatest overall mammary gland development and had the greatest PAR mass without compromised composition. When comparing EH- and R-fed calves given E2, differential expression of genes and pathways related to cell growth, cell signaling, and metabolism was observed. In summary, data indicates that enhanced feeding of calves in early life allows increased responsiveness to mammogenic stimuli and a corresponding increase in mammary development. We suggest that this may at least partly explain the improved future milk production in calves fed in this manner. / Ph. D.

The objective of this experiment was to examine the effects of varying the interval from follicular wave emergence to progestin removal on follicular dynamics and the synchrony of estrus. The experimental design was a 2x2x2 factorial with GnRH or estradiol-17 beta (E2) + progesterone (P4), controlled internal drug-releasing device (CIDR) treatment duration, and PG or saline treatment as main effects. Cycling, Angus cows (n=49), on d 6 to 8 of the estrous cycle, were randomly assigned to receive a CIDR treatment for 7 or 9 d. Approximately half of the cows from each CIDR group received either GnRH (100 mcg) or E2+P4 (1 mg E2 + 100 mg P4) at CIDR insertion. Cows in GnRH or E2+P4 groups were further divided into those that received PG (37.5 mg) or saline at CIDR insertion. All cows received PG (25 mg) 1 d prior to CIDR removal. The interval from follicular wave emergence to CIDR removal was longer for cows treated with GnRH (6.6 d) or a CIDR for 9 d (6.5 d) compared to those treated with E2+P4 (4.7 d) or a 7-d CIDR (4.8 d) (P < 0.05). Cows treated with PG or GnRH at CIDR insertion or a 9-d CIDR had a larger dominant follicle (DF) at CIDR removal than those treated with saline, E2+P4, or a 7-d CIDR. (P < 0.07). Altering the interval from wave emergence to progestin removal created differences in size of the DF at CIDR removal but did not affect the synchrony of estrus. / Master of Science

Natural steroid estrogens such as 17beta-estradiol in low nanogram per liter concentrations can adversely affect the reproductive health of aquatic organisms. The overall goal of this research was to quantify the sorption of 17beta-estradiol to three soils considered to be agriculturally productive from different physiographic regions in Virginia to aid in modeling the concentration of estrogens available for transport in runoff from agricultural fields. Batch equilibrium experiments were conducted with various concentrations of 17beta-estradiol (E2) in a background solution of 5 mM calcium chloride and 100 mg/L sodium azide added to four separate soil samples representative of productive agricultural soils from three different physiographic regions of Virginia. Groseclose loam, Myatt sandy loam and Cecil loam were supplied by the Crop and Soil Environmental Sciences Department at Virginia Tech. All soils were collected from the plow layer (0 to 15 cm) except for an additional Cecil soil sample from the Bt horizon. The concentration of E2 in the liquid phase was measured by gas chromatography/mass spectrometry (GC/MS) and was used to find the time to reach equilibrium and to develop sorption isotherms for each soil. The time required to reach equilibrium for all soils was less than 24 hours. A linear isotherm provided the best fit to model the sorption of E2 to Cecil and Myatt soils (R2 = 0.94 and 0.96, respectively). For Groseclose soil, the general form of the Freundlich isotherm fit best (R2 = 0.98), although the linear isotherm also provided a good fit (R2 = 0.93). The sorption of E2 to agricultural soil appears to be related to the organic carbon content of each soil (Pearson coefficient, 0.82). Attempts to analyze and create isotherms for conjugated E2 by deconjugating with metholysis were unsuccessful. / Master of Science

No / This work investigated the effect of electroporation on human epidermal penetration of a model neutral lipophilic compound (estradiol) from saturated aqueous solution and when encapsulated in ultradeformable liposomes. Total amount penetrated and skin deposition were compared with values obtained from passive diffusion. The effect of electrical pulsing on liposome size was investigated. The action of phosphatidylcholine on skin that was structurally altered by such pulses was determined. Electroporation did not affect liposome size. Skin pulsing considerably increased estradiol penetration and skin deposition from solution, relative to passive delivery, with subsequent partial recovery of skin resistance to molecular penetration. Surprisingly, with liposomes, electroporation did not markedly affect estradiol skin penetration. Importantly, liposomal phosphatidylcholine applied during or after pulsing accelerated skin barrier repair, i.e. provided an anti-enhancer or retardant effect.

No / Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24–48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin.—Pomari, E., Valle, L. D., Pertile, P., Colombo, L., and Thornton, M. J. Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

The antineoplastic drug doxorubicin is widely used in the treatment of various types of cancers including breast, colon and lung cancer. However, doxorubicin has adverse effects on the heart and prolonged doxorubicin administration results in cardiomyopathy and congestive heart failure. In the present study we have established that treatment of rat cardiac myoblasts (H9c2 cells) for 24 hours with doxorubicin resulted in concentration and time dependent cell death as determined by proliferation assay. Almost 50-55% cell death was attained at 24 hours treatment of H9c2 cells with 5 μM doxorubicin. We have selected about 50% cell injury as an optimum doxorubicin-induced cell injury because once this threshold is reached, cells became irreversibly injured and are unable to respond to protective treatment. We have observed that another potent antineoplastic drug, cyclophosphamide, had no cardiotoxic effects even with exposure at 35 μM concentrations for a treatment time of up to 72 hours. Pretreatment of H9c2 cells for 24 hours with 100 nM 17β-estradiol protects about 30% cell death against subsequent treatment for 24 hours with 5 μM doxorubicin. Interestingly 500 nM quecertin and 20 μM resveratrol pretreatment provide about 30% and 40% protection, respectively, to the H9c2 cells against subsequent doxorubicin treatment. However, diethylstilbestrol (DES), bisphenol A, and estrone exhibit no protective effects. It is concluded that 17β-estradiol, resveratrol and quercetin considerably protect H9c2 cells against doxorubicin-induced cell death.