The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for identifying novel estrogen response elements, to characterize estrogen receptor variants cloned from human breast tumors, and to examine the effect of estrogen receptor on the regulation of osteocalcin gene.

Cortisol, a hormone produced by a hormone produced by the adrenal gland, is responsible for many regulatory functions in the body. Cortisol release is mediated by adrenocorticotrophic hormone, or ACTH, through the hypothalamus-pituitary-adrenal or HPA axis. This HPA axis is the major release pathway used during acute stress, during which the levels of ACTH parallel those of cortisol. However, in states of chronic stress, the level of ACTH drops dramatically, while cortisol remains high. This study focuses on the pathway of cortisol release during these chronic stress states, specifically examining the role of IL-6 with respect to STATs and the Jak/STAT pathway. It has been shown that IL-6 increases cortisol levels, and that IL-6 utilizes the Jak/STAT pathway. Also, the steroidogenic acute regulatory (StAR) promoter contains multiple STAT binding sites. Thus, STATs could be mediating the effects of IL-6 in the chronic release of cortisol by inducing expression of StAR. Experiments were performed to identify whether IL-6 has a direct effect on StAR promoter activity, StAR mRNA and StAR protein levels. Electromobility Shift Assays (EMSA) were performed to show that STATs bind to the full STAT site within the StAR promoter region. Various experiments were also carried out in the presence of IL-6 alone or, congruently with either a Jak (AG490) or STAT3 (Piceatannol) inhibitor, to show the effects of STATs and the Jak/STAT pathway on StAR. Luciferase assays were performed in order to observe the effects on induction of the StAR promoter. RT-PCR and western blots were also performed to observe the effect of Jak/STAT inhibition on both StAR mRNA levels and StAR protein levels. These experiments showed a marked decrease in the IL-6-stimulated StAR promoter activity, mRNA and protein expression when treated with wither Jak or STAT inhibitor. Therefore, IL-6 regulates expression of StAR through utilization of the Jak/STAT pathway; which phosphorylates and subsequently dimerizes STAT, allowing STAT to translocate to the nucleus and bind to the StAR promoter, thus increasing StAR expression and thereby inducing synthesis of cortisol.

<p> Bisphenol-A (BPA), used in the production of polycarbonate plastics and epoxy resins, has established estrogenic properties. Early pregnancy in mice is highly sensitive to exogenous estrogens, particularly during the period of blastocyst implantation. Accordingly, I assessed pregnancy outcome, implantation, urinary hormone levels and uterine morphology following BPA exposure. Subcutaneous injections of BPA administered on days 1 through 4 of gestation reduced litter size at a dose of 3 mg/animal/day and decreased the proportion of parturient females at 10 mg/animal/day. Hysterectomies performed on day 6 of pregnancy confirmed a significant disruption of implantation occurring at doses as low as 6 mg/animal/day. Urinary progesterone levels were also reduced by 10 mg/animal/day. Uterine luminal area expanded substantially in response to increasing doses of BPA. Luminal epithelial cell height increased following exposure to 10.125 mg/animal, whereas there were no differences in the number of corpora lutea among conditions. The proportion of cells staining positively for estrogen receptors was affected non-monotonically, showing highest levels at 3.375 mg/animal and lowest levels at 10.125 mg/animal. Similarly progesterone receptor expression measured through western blots related non-monotonically to dose, being highest at 3.375 mg/animal and diminishing with increasing dose. Effects of a single administration of BPA on days 0, 1, or 2 of gestation were also investigated. A single dose of 10 mg reduced the number of implantation sites when given on day 0 or 1, and 6 mg did so on day 1, but there was no such effect of any dose administered on day 2. Exposure to low, environmentally-relevant doses of BPA did not result in any clear reproductive or hormonal effects. These studies highlight the detrimental effects BPA exposure induces during early pregnancy and provides further evidence of its weak estrogenic properties in vivo.</p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Tesis por compendio / [EN] During the last 25 years, eel populations have declined considerably, this species is considered outside safe biological limits. The life cycle of the eel is quite complex including transoceanic migration whose conditions are still so unknown that even the natural spawning process has never been observed. Eels cease feeding during migration and that is why their energy reserves such as lipids play a crucial role and thus their in deep study is essential to obtain gametes (eggs and sperm) quality as the first step for their reproduction in captivity. In the last years, many efforts have been directed at the study of the influence of fatty acids on the reproductive performance in females, but not in male eels. The present manuscript describes the results obtained in two experiments. The first experiment (which led to the three first articles included in the memory) focused on the effect of fatty acids along maturation, their relationship with steroid hormones and their effect on sperm quality parameters. The second experiment involved the comparison of different diets designed, considering the previously acquired insights into the most influential fatty acids on male eel reproduction, and the assessment of their influence on the reproductive performance of males. The first experiment was used to assess the changes in the fat content, as well as fatty acids, in different tissues of male eels hormonally induced to sexual maturation under different thermal regimes (two variables and one constant). Males finished spermatogenesis earlier with a constant temperature of 20 ºC, suggesting that eel spermatogenesis is tightly regulated by temperature. The fat content did not change significantly in the muscle, but was increased in the liver and decreased in testes during testicular development. With regard to fatty acids, during sperm maturation, the liver was highlighted as the main site of synthesis. Finally, EPA, ARA and DHA remained constant in testes, while the level of the rest of fatty acids decreased significantly. Using samples from the same experiment, the correlation between the main steroid hormones and fatty acids at different stages of testicular development were investigated. Similarly as occurs in mammals, EPA and DHA were highlighted as possible modulators of androgen synthesis. The set of the results obtained suggests new perspectives concerning the functions and interactions between fatty acids and steroids in fish spermatogenesis. Also, with samples from the first experiment, a study was carried out seeking to establish the possible correlations between different fatty acids and several sperm quality parameters. Correlations between several highly unsaturated fatty acids with parameters such as volume, the percentage of motile sperm and sperm velocity were found. Finally, with the acquired knowledge so far, a second experiment was carried out designing feeds with different fatty acid percentages in order to evaluate the influence of the diets on sperm quality. The results showed that high levels of DHA and EPA in the diets induce higher volumes and high sperm motility. This study allowed obtaining results applicable to the design of optimum broodstock diets for this species. / [ES] Durante los últimos 25 años las poblaciones de anguila han disminuido considerablemente, de modo que la especie se considera fuera de los límites biológicos de seguridad. El ciclo vital de la anguila es bastante complejo incluyendo una migración transoceánica cuyas condiciones son todavía tan desconocidas que incluso el proceso natural de desove no se ha observado nunca. Las anguilas dejan de alimentarse durante la migración y es por ello que sus reservas energéticas en forma de lípidos tienen un papel crucial y su estudio en profundidad es importante para obtener gametos (huevos y esperma) de calidad como primer paso para su reproducción en cautividad. En los últimos años se han dirigido muchos esfuerzos al estudio de la influencia de los ácidos grasos en el rendimiento reproductivo de las hembras, pero no en el de los machos de anguila. El presente documento describe los resultados obtenidos en dos experimentos. El primer experimento (que dio lugar a los tres primeros artículos incluidos en la memoria) se centró en el efecto de los ácidos grasos durante la maduración, su relación con las hormonas esteroideas y su efecto sobre los parámetros de calidad espermática. El segundo experimento consistió en la comparación de diferentes dietas, diseñadas considerando los conocimientos adquiridos previamente sobre los ácidos grasos más influyentes en la reproducción de machos de anguila, y en la evaluación de su influencia en el rendimiento reproductivo de los machos. El primer experimento sirvió para evaluar los cambios en el contenido graso, así como en los ácidos grasos, en diferentes tejidos de machos de anguila hormonalmente inducidos a la maduración sexual bajo diferentes regímenes térmicos (dos variables y uno constante). Los machos concluyeron la espermatogénesis más temprano a una temperatura contante de 20 ºC, sugiriendo que la espermatogénesis de anguila está estrechamente regulada por la temperatura. El contenido en grasa no varió significativamente en el músculo, se incrementó en el hígado y bajó en el testículo a lo largo del desarrollo testicular. Con respecto a los ácidos grasos, durante la maduración del esperma, el hígado destacó como el principal sitio de síntesis. Finalmente, EPA, ARA y DHA permanecieron constantes en el testículo, mientras que el nivel del resto de ácidos grasos bajó significativamente. Usando muestras del mismo experimento, se investigaron las correlaciones entre las principales hormonas esteroideas y los ácidos grasos en los diferentes estadios del desarrollo testicular. De forma similar a lo que ocurre en mamíferos, EPA y DHA destacaron como posibles moduladores de la síntesis de andrógenos. El conjunto de los resultados obtenidos sugiere nuevas perspectivas sobre las funciones y las interacciones entre los ácidos grasos y los esteroides en la espermatogénesis de peces. También con muestras del primer experimento, se llevó a cabo un estudio que buscó las posibles correlaciones de los diferentes ácidos grasos con varios parámetros de calidad espermática. Se encontraron correlaciones entre varios ácidos grasos altamente insaturados con parámetros como el volumen, el porcentaje de espermatozoides móviles y la velocidad del esperma. Finalmente, con los conocimientos adquiridos hasta el momento, se realizó un segundo experimento para el que se diseñaron piensos con diferentes porcentajes de ácidos grasos con el fin de evaluar la influencia de las dietas sobre la calidad del esperma. Los resultados mostraron que niveles altos de DHA y EPA en las dietas inducen mayores volúmenes y una alta motilidad del esperma. Este estudio permitió obtener resultados aplicables al diseño de dietas óptimas para reproductores de esta especie. / [CA] Durant els últims 25 anys les poblacions d'anguila han disminuït considerablement, de manera que l'espècie es considera fora dels límits biològics de seguretat. El cicle vital de l'anguila és bastant complex incloint una migració transoceànica les condicions de la qual són encara tan desconegudes que fins i tot el procés natural de fresa mai ha estat observat. Les anguiles deixen d'alimentar-se durant la migració i és per això que les seves reserves energètiques en forma de lípids tenen un paper crucial i el seu estudi en profunditat és important per a obtenir gamets (ous i esperma) de qualitat com a primer pas per a la reproducció en captivitat. En els últims anys s'han dirigit molts esforços a l'estudi de la influència dels àcids grassos en el rendiment reproductiu de les femelles, però no en el dels mascles d'anguila. El present document descriu els resultats obtinguts en dos experiments. El primer (que dona lloc als tres primers articles inclosos en la memòria) es va centrar en l'efecte dels àcids grassos durant la maduració, la seva relació amb les hormones esteroides i el seu efecte sobre els paràmetres de qualitat espermàtica. El segon experiment va consistir en la comparació de diferents dietes, dissenyades considerant els coneixements adquirits prèviament sobre els àcids grasos més influents en la reproducció dels mascles d'anguila, i en l'avaluació de la seva influència en el rendiment reproductiu dels mascles. El primer experiment va servir per avaluar els canvis en el contingut de gras, així com en els àcids grassos, en els diferents teixits de mascles d'anguila hormonalment induïts a la maduració sexual baix diferents règims tèrmics (dos variables i un constant). Els mascles van concloure la espermatogènesis més prompte a una temperatura de 20 ºC, suggerint que l'espermatogènesis d'anguila està estretament regulada per la temperatura. El contingut en greix, no va variar significativament en el múscul, es va incrementar en el fetge i va baixar en els testicles al llarg del desenvolupament testicular. Pel que fa als àcids grassos, durant la maduració de l'esperma, el fetge va destacar com al principal lloc de síntesi. Finalment, EPA, ARA y DHA van romandre constants en el testicle, mentre que el nivell de la resta d'àcids grassos va baixar significativament. Emprant mostres del mateix experiment, es van investigar les correlacions entre les principals hormones esteroides i els àcids grassos en els diferents estadis de desenvolupament testicular. De manera semblant al que ocorre en mamífers, EPA i DHA van destacar com a possibles moduladors de la síntesi d'andrògens. El conjunt dels resultats obtinguts suggerixen noves prespectives sobre les funcions i les interaccions entre els àcids grassos i els esteroides en l'espermatogènesi dels peixos. També amb mostres del primer experiment, es va dur a terme un estudi que va buscar les possibles correlacions dels diferents àcids grassos amb diversos paràmetres de qualitat espermàtica. Es van trobar correlacions entre diversos àcids grassos altament insaturats amb paràmetres com el volum, el percentatge d'espermatozoides mòbils i la velocitat de l'esperma. Finalment, amb els coneixements adquirits fins al moment, es va realitzar un experiment per al qual es van dissenyar pinsos mb diferents percentatges d'àcids grassos per tal d'avaluar la influència de les dietes sobre la qualitat de l'esperma. Els resultats van mostrar que nivells alts de DHA i EPA en les dietes indueixen a majors volums i una alta motilitat de l'esperma. Aquest estudi va permetre obtenir resultats aplicables al disseny de dietes òptimes per a reproductors d'aquesta espècie. / Baeza Ariño, R. (2015). Roles of lipids and fatty acids through the spermatogenesis of European eel (Anguilla anguilla) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/49988 / Compendio

No description available.

We investigated the gestational expression of androgen receptor (AR) and defined its regulation and that of its co-repressors, PSF and p54nrb, by steroid hormones and myometrial stretch in vivo in pregnant and non-pregnant rats. Our data demonstrate that, 1) myometrial AR expression decreases prior to term; 2) AR expression is up-regulated by MPA treatment and down-regulated by mechanical stretch; (3) myometrial PSF protein expression is down-regulated by estrogen signaling and by mechanical stretch, and up-regulated by androgen signaling; (4) while myometrial PSF mRNA expression is also down-regulated by stretch, the regulation by estrogen and P4 on PSF mRNA appear to be opposite to the effects on PSF protein. We conclude that the decreased androgen signaling in late pregnancy (as a result of decreased AR and PSF expression mediated by hormonal and mechanical signals) may contribute to the mechanisms leading to labor initiation.

We investigated the gestational expression of androgen receptor (AR) and defined its regulation and that of its co-repressors, PSF and p54nrb, by steroid hormones and myometrial stretch in vivo in pregnant and non-pregnant rats. Our data demonstrate that, 1) myometrial AR expression decreases prior to term; 2) AR expression is up-regulated by MPA treatment and down-regulated by mechanical stretch; (3) myometrial PSF protein expression is down-regulated by estrogen signaling and by mechanical stretch, and up-regulated by androgen signaling; (4) while myometrial PSF mRNA expression is also down-regulated by stretch, the regulation by estrogen and P4 on PSF mRNA appear to be opposite to the effects on PSF protein. We conclude that the decreased androgen signaling in late pregnancy (as a result of decreased AR and PSF expression mediated by hormonal and mechanical signals) may contribute to the mechanisms leading to labor initiation.

No / Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).